Laboratory Medicine Diagnosis of Disease in Clinical Laboratory

Laboratory Medicine
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a LANGE medical book
Laboratory Medicine
The Diagnosis of Disease
in the Clinical Laboratory
Second Edition
Edited by
Michael Laposata, MD, PhD
Chairman
Department of Pathology
University of Texas Medical Branch
Galveston, Texas
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To Susan, with love
Key Features of
Laboratory Medicine
A complete full-color guide to selecting the correct
laboratory test and accurately interpreting the
results—covering the entire field of clinical pathology
• 46 laboratory methods presented in easy-to-understand illustrations which include
information on the expense and complexity of the assays
• Features an easy-to-follow, consistent presentation for each disease discussed
• More than 200 tables and full-color algorithms encapsulate important information
and facilitate understanding
• Full-color blood-smear micrographs demonstrate common abnormal morphologies
of red blood cells
• Valuable learning aids in each chapter,
including learning objectives, chapter CHAPTER 10 Diseases of Red Blood Cells 231
outlines, and a general introduction

• Extensive table of Clinical Laboratory
Reference Values showing the conversions
between U.S. and SI units for each value
• An essential text for medical students
and residents studying clinical pathology,
medical technology students, and for
practitioners working in a clinical setting FIGURE 10–15 Peripheral blood smear from a
patient with large numbers of elliptocytes.
FIGURE 10–16 A peripheral blood smear stained with
Wright stain showing reticulocytes.
• This edition has been enhanced by coverage

of genetic test options that are now
commonly used in clinical practice.
Blood-smear micrographs demonstrate common

abnormal morphologies of red blood cells
FIGURE 10–17 A peripheral blood smear showing FIGURE 10–18 A peripheral blood smear from a patient
circulating nucleated red blood cells (arrowheads), as with stomatocytes.
well as Howell–Jolly bodies (arrows).
CHAPTER 22 The Endocrine System 423
[–]
Hypothalamus
Thyrotropin-releasing
hormone (TRH)
[+]
[–]
Pituitary
Thyroid-stimulating
T3 200 tables and full-color algorithms
hormone (TSH) T4
[+]
Thyroid
rT3
T3 is primary
feedback stimulus
encapsulate important information
FIGURE 22–2 Hypothalamic–pituitary–thyroid interactions. [+] Stimulation; [−] inhibition.
426 CHAPTER 22 The Endocrine System

hormone replacement in hypothyroid patients. Third-generation assays are essential for monitor-
ing TSH suppression therapy in patients with a TSH-responsive thyroid tumor.
The relationship between TSH and the thyroid hormones, particularly free T4, is an inverse
log-linear one, such that very small changes in free T4 result in large changes in TSH. Thus, TSH TABLE 22–1 Laboratory Evaluation of Patients for Thyroid Disease
is the most sensitive first-line screening test for suspected thyroid abnormalities. If the TSH is Laboratory Test Results Suggestive of Diagnosis in the Appropriate
Disorder Clinical Setting
within the normal reference range, no further testing is performed. If the TSH is outside of the
reference range, a free T4 is obtained. Hyperthyroidism
Graves disease TSH low; free T4 high; in some cases, T3 is elevated and free T4 is normal;
TRAbs or TSI elevated
Uptake into cells Toxic multinodular goiter TSH low; free T4 and T3 normal or high; normal or increased radioactive iodine
uptake; thyroid scan with multiple areas of increased uptake surrounded by
Oral intake Plasma producing thyroid homone Thyroidal suppressed uptake
of iodine iodide iodide
Toxic adenoma TSH low; free T4 and T3 normal or high; normal or increased radioactive iodine
uptake; thyroid scan with focal increased uptake in tumor surrounded by
Binding of iodide suppressed uptake in nontumor tissue
to thyroglobulin Subacute thyroiditis TSH low; free T4 and T3 high; increased; decreased radioactive iodine uptake
Painless thyroiditis TSH low; free T4 and T3 high; erythrocyte sedimentation rate normal;
Coupling of Monoiodotyrosine decreased radioactive iodine uptake
Proteases Thyroglobulin
iodotyrosines (MIT) and
Free T4 release T3 bound Hypothyroidism
diiodotyrosine
and T4 from T3 and T4 (DIT) residues on Hashimoto thyroiditis TSH high; T4 normal and then low, preceding a decline in T3; anti-TPO and/or
thyroglobulin
thyroglobulin antithyroglobulin antibody positive
Ablative hypothyroidism TSH high; free T4 and T3 low following procedure that ablates thyroid
Deiodination
Infantile hypothyroidism TSH high; free T4 low in a newborn or infant

Free T3 Thyroid hormones Euthyroid sick syndrome TSH normal to high; free T4 normal; T3 low; rT3 high; concentrations of TSH and
transported to tissues thyroid hormones vary throughout disease course
on thyroid-binding
rT3, reverse triiodothyronine; T3, triiodothyronine; free T4, free thyroxine; TSH, thyroid-stimulating hormone; TRAbs, TSH receptor
globulin (TBG), transthyretin, autoantibodies; TSI, thyroid-stimulating immunoglobulins.
Reverse and albumin Diverse metabolic
T3 (rT3) effects on target cells
may have exophthalmos, emotional changes, menstrual changes, and a fine tremor of the hands.
In the presence of a clinical history and physical examination consistent with hyperthyroidism,
Free T4
a diagnosis of hyperthyroidism (but not necessarily its cause) can be established by the demon-
stration of a low TSH level and a high free T4. In uncommon situations, only the total T3 level
FIGURE 22–3 The formation, secretion, and transport of thyroid hormones. is elevated and the serum free T4 is normal (T3 thyrotoxicosis). To determine the etiology of the
hyperthyroidism, additional testing is usually necessary. Graves disease, toxic multinodular goiter
(TMNG), and toxic adenoma account for the vast majority (>95%) of cases of hyperthyroidism. It
should be noted that diffuse or focal enlargement of the thyroid gland, also known as goiter, can
be associated with hyperfunction, normal function, and hypofunction of the gland.
Thyroid Storm
Thyroid storm is a relatively uncommon, but life-threatening manifestation of hyperthyroid-
ism caused by excess circulation of thyroid hormones. Symptoms of thyroid storm are similar,
Graves disease is a but much more severe than traditional hyperthyroidism, including a markedly high fever of
relatively common 105°F to 106°F, tachycardia, hypertension, and neurological and gastrointestinal abnormali-
hyperthyroid disorder ties. Thyroid storm is precipitated by acute illnesses such as sepsis, diabetic ketoacidosis, and
occurring more preeclampsia, as well as surgical or other diagnostic or therapeutic actions such as radioac-
frequently in women. tive iodine use, anesthesia, excessive thyroid hormone ingestion, or thyroid palpation. Thyroid
It is an autoimmune storm is associated with a high fatality rate if not identifi ed early. The diagnosis is based on the
disease caused by TSH presence of clinical signs and symptoms of severe hyperthyroidism in the context of a precipi-
receptor autoantibodies tating cause. In addition, marked elevations in free and total T4 are common in thyroid storm.
that bind to and stimulate Total T3 is unreliable in this setting because concomitant nonthyroidal illness (NTI) may cause
TSH receptors resulting in
T3 to decrease significantly.
autonomous production
of thyroid hormone.
Graves Disease
Graves disease is a relatively common hyperthyroid disorder occurring more frequently in
women. It has a familial predisposition. It is an autoimmune disease caused by TSH receptor
C H A P T E R
Breast
Karin E. Finberg 21
LEARNING OBJECTIVES
1. Learn the tissue- and serum-based biomarkers in breast cancer.
2. Understand how the individual biomarkers are used clinically.
CHAPTER OUTLINE
Introduction 413 Hereditary Breast and Ovarian
Breast Cancer 413 Cancer Syndrome 417
Laboratory Testing 414 Other High-penetrance Cancer
Tissue-based Biomarkers Predisposition Genes 418
in Breast Cancer 414 Li-Fraumeni Syndrome 418
Serum-based Biomarkers Cowden Syndrome 418
in Breast Cancer 416 Peutz-Jeghers Syndrome 419
INTRODUCTION
This chapter focuses on laboratory testing relevant to breast cancer. Infections of the breast are
included in Chapter 5.
BREAST CANCER
Description
Cancers of the breast constitute a major cause of mortality in women of Western countries. In the
United States, the lifetime probability that a woman will develop breast cancer is 1 in 8. Breast
cancer accounts for 29% of new cancer cases and 14% of cancer deaths in American women.
About 1% of breast cancers occur in males. The risk of developing breast cancer is influenced by
several factors. These factors include increased age, family history of breast cancer (especially in
a first-degree relative), hormonal factors (early age at menarche, older age of menopause, older
age at first full-term pregnancy, fewer number of pregnancies, and use of hormone replacement
therapy), clinical factors (high breast tissue density and benign breast diseases associated with
atypical hyperplasia), obesity, and alcohol consumption. Since 1990, the mortality rate associated
Valuable learning aids are
with female breast cancer has decreased in the United States, a decline that has been attributed to
both therapeutic advances and early detection.
For localized breast cancer, primary treatment typically consists of either breast-conserving
surgery and radiation or mastectomy. Most patients with invasive breast cancer subsequently
included in each chapter
receive systemic adjuvant chemotherapy and/or hormone therapy, both of which have been
shown to reduce systemic recurrence and breast cancer-related mortality. However, the fact
that some patients who lack lymph node involvement are cured by the combination of sur-
gery and radiotherapy suggests that adjuvant treatment may not be necessary in all cases.
413
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Contents
Authors xi
Preface xiii
Acknowledgments xv
Clinical Laboratory Reference Values xvii
1. Concepts in Laboratory Medicine 1 13. Diseases of White Blood Cells,

Michael Laposata, MD, PhD Lymph Nodes, and Spleen 317
Daniel E. Sabath, MD, PhD
2. Methods 13
Michael Laposata, MD, PhD, 14. The Respiratory System 339
James H. Nichols, PhD Alison Woodworth, PhD
Paul Steele, MD, & Thomas P. Stricker, MD, PhD 15. The Gastrointestinal Tract 351
3. Autoimmune Disorders Involving the Michael Laposata, MD, PhD &
Connective Tissue and Immunodeficiency D. Robert Dufour, MD, FCAP, FACB
Diseases 61 16. The Liver and Biliary Tract 357
Mandakolathur R. Murali, MD William E. Winter, MD
4. Histocompatibility Testing 17. Pancreatic Disorders 371
and Transplantation 83 David N. Alter, MD &
Yash P. Agrawal, MD, PhD & Michael Laposata, MD, PhD
Susan L. Saidman, PhD
18. The Kidney 385
5. Infectious Diseases 91 William E. Winter, MD
Eric D. Spitzer, MD, PhD
19. Male Genital Tract 397
6. Toxicology 159 Mark H. Wener, MD, ABIM, ABAI (CLI/DLI),
James H. Nichols, PhD, Sheila P. Dawling, PhD, Charles H. Muller, PhD, HCLD (AAB),
& Michael Laposata, MD, PhD & D. Robert Dufour, MD, FCAP, FACB
7. Diseases of Infancy and Childhood 185 20. Female Genital System 405
Paul Steele, MD Stacy E.F. Melanson, MD, PhD &
8. Blood Vessels 197 Ann M. Gronowski, PhD
Michael Laposata, MD, PhD 21. Breast 413
9. The Heart 209 Karin E. Finberg, MD, PhD
Fred S. Apple, PhD 22. The Endocrine System 421
10. Diseases of Red Blood Cells 221 Alison Woodworth, PhD, Vipul Lakhani, MD,
Daniel D. Mais, MD Samir L. Aleryani, PhD, C(ASCP), CLS (NCA),
FACB, & Michael Laposata, MD, PhD
11. Bleeding and Thrombotic Disorders 253
Elizabeth M. Van Cott, MD & Index 469
12. Transfusion Medicine 291
Christopher P. Stowell, MD, PhD &
Jacqueline J. Haas, MD
ix
Authors
Yash P. Agrawal, MD, PhD Jacqueline J. Haas, MD

Professor of Clinical Pathology and Laboratory Staff Pathologist, Laboratory Medical Director,
Medicine, Department of Pathology and Laboratory Department of Pathology, Clinical Pathology Associates,
Medicine; Director of Central Laboratory; Director of St. David’s Medical Center, Austin, Texas
Point of Care Testing Services, New York Presbyterian
Vipul Lakhani, MD
Hospital-Cornell Campus, New York, New York
Assistant Professor of Medicine, Department of
Samir L. Aleryani, PhD, C(ASCP), CLS (NCA), FACB Medicine, Division of Endocrinology, Vanderbilt
Formerly Assistant Professor in Clinical Pathology, University Medical Center, Nashville, Tennessee
Director Clinical Research Center Laboratory, Vanderbilt
Institute of Clinical and Translational Research
Chairman, Department of Pathology, University of Texas
(VICTR), Department of Pathology, Microbiology, and
Medical Branch, Galveston, Texas
Immunology School of Medicine, Vanderbilt University,
Nashville, Tennessee Daniel D. Mais, MD
Clinical Pathology Associates, Department of Pathology,
David N. Alter, MD
Baptist Health System, San Antonio, Texas
Clinical/Chemical Pathologist, Spectrum Health/
Michigan Pathology Specialists, Grand Rapids, Michigan Stacy E.F. Melanson, MD, PhD
Assistant Professor, Associate Medical Director,
Fred S. Apple, PhD
Clinical Chemistry, Clinical Laboratories, Harvard
Medical Director, Clinical Laboratories, Hennepin
Medical School Brigham and Women’s Hospital,
County Medical Center; Professor, Laboratory Medicine
Boston, Massachusetts
and Pathology, University of Minnesota School of
Medicine, Minneapolis, Minnesota Charles H. Muller, PhD, HCLD (AAB)
Director, Male Fertility Laboratory and Lecturer,
Sheila P. Dawling, PhD
Full-Time, Department of Urology and Biological
Vice President of Laboratories, Aegis Sciences
Structure, University of Washington School of Medicine,
Corporation, Nashville, Tennessee
Seattle, Washington
D. Robert Dufour, MD, FCAP, FACB
Mandakolathur R. Murali, MD
Consultant, Emeritus Professor of Pathology,
Director, Assistant Professor, Department of Clinical
Department of Pathology and Hepatology, Veterans
Immunology Laboratory, Massachusetts General
Affairs Medical Center, The George Washington
Hospital, Harvard Medical School, Boston, Massachusetts
University Medical, Washington, District of Columbia
James H. Nichols, PhD
Karin E. Finberg, MD, PhD
Professor of Pathology, Microbiology and Immunology;
Assistant Professor of Pathology, Yale School of
Medical Director of Clinical Chemistry, Vanderbilt
Medicine, New Haven, Connecticut
University School of Medicine, Nashville, Tennessee
Ann M. Gronowski, PhD
Professor, Department of Pathology & Immunology and
Obstetrics & Gynecology, Washington University School
of Medicine, St. Louis, Missouri
xi
xii AUTHORS
Daniel E. Sabath, MD, PhD Thomas P. Stricker, MD, PhD

Associate Professor, Department of Laboratory Assistant Professor, Department of Pathology,
Medicine; Adjunct Associate Professor, Department Microbiology, and Immunology, Vanderbilt University
of Medicine (Medical Genetics); Head, Hematology School of Medicine, Nashville, Tennessee
Division, Department of Laboratory Medicine; Director
Elizabeth M. Van Cott, MD
of Hematology Laboratories, University of Washington
Director, Coagulation Laboratory; Medical Director,
Medical Center and Harborview Medical Center,
Core Laboratory, Massachusetts General Hospital;
Seattle, Washington
Associate Professor, Harvard Medical School,
Susan L. Saidman, PhD Boston, Massachusetts
Associate Professor of Pathology, Harvard Medical
Mark H. Wener, MD, ABIM, ABAI (CLI/DLI)
School; Director, Histocompatibility Laboratory,
Professor, Departments of Laboratory Medicine &
Massachusetts General Hospital, Boston, Massachusetts
Medicine, University of Washington, Seattle, Washington
Eric D. Spitzer, MD, PhD
William E. Winter, MD
Associate Professor, Department of Pathology, Stony
Professor, Department of Pathology, Immunology, and
Brook University Medical Center, Stony Brook,
Laboratory Medicinem, University of Florida College of
New York
Medicine, Gainesville, Florida
Paul Steele, MD
Alison Woodworth, PhD
Medical Director, Clinical Associate Professor,
Assistant Professor, Medical Director of Esoteric
Department of Pathology and Laboratory Medicine,
Chemistry, Department of Pathology, Microbiology, and
Clinical Laboratories, Cincinnati Children’s
Immunology, Vanderbilt University Medical Center,
Hospital Medical Center, University of Cincinnati,
Nashville, Tennessee
Cincinnati, Ohio
Christopher P. Stowell, MD, PhD
Director, Blood Transfusion Service; Associate Professor
of Pathology, Department of Pathology, Massachusetts
General Hospital, Harvard Medical School,
Boston, Massachusetts
Preface
In the early 1990s when I was at the Massachusetts General test results by the physicians who ordered the tests is incor-
Hospital as director of clinical laboratories, I was invited by rect. A survey of medical schools currently underway has
Ramzi Cotran to join him and Stan Robbins at the Brigham shown that the teaching of laboratory medicine over the full
and Women’s Hospital for a meeting. In that meeting, they 4 years of medical school includes (as a mean value across
indicated to me that the Robbins Pathologic Basis of Disease, the US medical schools) only about 10 hours of formal train-
primarily an anatomic pathology book, would greatly benefit ing in laboratory medicine. This study also shows that, unlike
from a parallel book in clinical pathology (laboratory medi- virtually every other medical discipline, laboratory medicine
cine). At that time, areas such as coagulation and toxicology is commonly not taught by experts in the field, even if they
were expanding rapidly with new disorders and new tests to are present in the institution. As a result, medical schools
diagnose them. Because there was little anatomic pathology graduate physicians who have had almost no training in
in these fields, the discussions of these major areas of diag- something they do virtually every day—order laboratory
nostic medicine in the Robbins book were limited. In addi- tests and interpret the test results. Surprisingly, the patients
tion, as the test menu in the clinical laboratory was growing and the medical institutions suffer cost and care disadvan-
in complexity and cost, many important clinical laboratory tages quietly and unknowingly. There are surely hundreds of
tests for common disorders, such as the troponin test for patients every month in the United States who present to an
myocardial infarction, were also discussed only briefly in the emergency room with shortness of breath, for whom a diag-
Robbins book. Both Robbins and Cotran understood that nosis of pulmonary embolism is overlooked, and an appro-
a discussion regarding the threshold for diagnosis of myo- priate test (the D-dimer test for pulmonary embolism) is not
cardial infarction, as troponin testing rapidly evolved and ordered. Such patients are discharged from the emergency
improved, was necessary to fully discuss the topic. There room without ever being anticoagulated, and for some, to
were many twists and turns from that meeting about 20 years die shortly thereafter, from an expansion of the pulmonary
ago to the development of this second edition of Laboratory embolism. Like surgical errors or medication errors, the
Medicine: The Diagnosis of Disease in the Clinical Laboratory error of the healthcare provider who did not order a neces-
in the prestigious Lange series by McGraw-Hill. With this sary test results in a preventable death—but unlike surgical
second edition, I believe we truly have a book that is essen- and medication errors, the fact that such a case represents
tial for education of medical students and residents study- a preventable death is rarely recognized by the patient, the
ing clinical pathology, and importantly, for practitioners in patient’s family, fellow physicians, and often even the physi-
a clinical setting. By selecting the correct tests and interpret- cian who failed to order the correct test.
ing the results correctly, physicians using this book should There are several groups of healthcare providers who
be able to optimize patient outcomes and reduce the cost to would benefit significantly by using this book to correctly
achieve a diagnosis. order laboratory tests and correctly interpret the test results.
This second edition is a great step forward from the first Certainly, there is every reason to believe that medical stu-
edition. It contains information about genetic tests now in dents can learn the histopathologic changes associated with a
common use. Additional descriptions of test methods with disease using a textbook such as the Robbins Pathologic Basis
simply illustrated figures have been added to this edition. of Disease, and learn laboratory tests associated with that dis-
The authors of the individual chapters have all taken sig- order, using this book, at the same time.
nificant steps to make the tables that indicate the diagnos- Medical technology students would greatly benefit by a
tic tests for different clinical conditions more concise and thorough understanding of the methods that are illustrated in
easy to understand. It is now clear that significant morbidity Chapter 2 of this book. In addition, it would be of immense
and mortality occur on a daily basis, affecting thousands of benefit for medical technology students to more fully under-
patients, because incorrect tests are ordered, important tests stand the clinical significance of the test results that they
for the diagnosis are omitted, and/or the interpretation of generate so that they can more knowledgeably interact with
xiii
xiv PREFACE
physicians who are confused about laboratory test results. order, and importantly, how to interpret the result as well by
Interactions between medical technologists and physicians describing common interpretation mistakes – with a much
ordering tests that result in improved performance in test higher reliability than virtually all of what is available on the
selection and result interpretation would greatly increase the Internet.
respect for the medical technologist (also known as clinical It is my greatest hope that the use of this textbook,
laboratory scientist) from physicians who use the clinical which presents the entire field of laboratory medicine to a
laboratory. large audience of future physicians, medical technologists,
In conversations with primary care physicians attempt- and healthcare providers ordering laboratory tests, will
ing to select the correct laboratory tests, they often indicate result in better clinical outcomes for patients at a greatly
that one of their first inquiries about which laboratory tests reduced cost.
to select is to search Wikipedia. It is most likely that there is
Michael Laposata
a table in this textbook, written by a prominent expert in the
Galveston, Texas
field, that will tell a practicing physician exactly what test to
Acknowledgments
I would first like to acknowledge all the expert chapter authors of the second edition. They are both effective editors.
associated with this textbook. Many of them have been I would also like to extend my deepest thanks to the others
close professional friends for many years, and I am deeply at McGraw-Hill who have been involved in the production
honored to be a colleague of theirs. I also worked closely with of this book. I am delighted that this book has been included
Mr. Robert Pancotti at McGraw-Hill in the production of the in the Lange series of medical books, which has such a proud
first edition of the book, and Ms. Cindy Yoo in production tradition in medical education.
xv
Clinical Laboratory Reference Values
The conventional units in this table are the ones most com- samples and serum samples are unacceptable. For other
monly used in the United States. Outside the United States, compounds, both plasma samples and serum samples may
SI units are the predominant nomenclature for laboratory test be acceptable. However, there may be differences, often
results. The base units in the SI system related to laboratory minor, in the results obtained using plasma versus serum.
testing that are found in this table include the mole (amount Potassium is 1 such compound in which reference ranges
of substance), meter (length), kilogram (mass), second (time), may be different for plasma and serum. There is a signifi-
and Celsius (temperature). cant movement away from the use of serum in favor of
Reference ranges vary depending on the instrument and plasma. The principal reason for this is that extra time is
the reagents used to perform the test. Therefore, the reference required for samples to clot so that serum may be generated.
ranges shown in this table are only close approximations to the A sample collected into a tube with anticoagulant results in
adult reference ranges found in an individual clinical labora- the generation of plasma rather than serum after the tube
tory. It is also important to understand that reference ranges is centrifuged. The clotting step is omitted when plasma
can be significantly affected by age and sex. samples are prepared, and therefore the turnaround time for
Conversion factors are provided in the table to allow the the performance of the test is shortened. In some circum-
reader to convert conventional units to SI units and vice versa. stances, whole blood is used for analysis, but the number
The conversion of the conventional unit to SI unit requires a of tests performed using whole blood is very limited. Urine
multiplication with the conversion factor, and conversion of and other body fluids, such as pleural fluid and cerebrospi-
the SI unit to the conventional unit requires division by the nal fluid, are also used for testing. Some of the entries in the
conversion factor. table are associated with a fluid other than plasma, serum,
The sample fluid is sometimes highly restrictive. For or whole blood.
example, coagulation tests must be performed using plasma
xvii
xviii CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
Reference Traditional Multiply →, SI Reference
Specimen Interval Units ← Divide Interval SI Units
Acetaminophen (therapeutic) Serum, plasma 10-30 μg/mL 6.62 70-200 μmol/L
Acetoacetic acid Serum, plasma <1 mg/dL 0.098 <0.1 mmol/L
Acetone Serum, plasma <2.0 mg/dL 0.172 <0.34 mmol/L
Acetylcholinesterase Red blood cells 5-10 U/mL 1 5-10 U/L
Activated partial thromboplastin Whole blood 25-40 s 1 25-40 s

time (APTT)
Adenosine deaminasea Serum 11.5-25.0 U/L 0.017 0.20-0.43 μKat/L
Adrenocorticotropic hormone (ACTH) (see corticotropin)
Alanineb (adult) Plasma 1.87-5.88 mg/dL 112.2 210-661 μmol/day
Alanine aminotransferase Serum 10-40 U/L 1 10-40 U/L

(ALT, SGPT)b
Albuminb Serum 3.5-5.0 g/dL 10 35-50 g/L
Alcohol (see ethanol, isopropanol, methanol)
Alcohol dehydrogenasea Serum <2.8 U/L 0.017 <0.05 μKat/L
Aldolasea,b Serum 1.0-7.5 U/L 0.017 0.02-0.13 μKat/L
Aldosteroneb (upright) Plasma 7-30 ng/dL 0.0277 0.19-0.83 nmol/L
Aldosterone Urine, 24 h 3-20 μg/24 h 2.77 8-55 nmol/day
Alkaline phosphataseb Serum 50-120 U/L 1 50-120 U/L
α1-Acid glycoprotein Serum 50-120 mg/dL 0.01 0.5-1.2 g/L
α2-Macroglobulin Serum 130-300 mg/dL 0.01 1.3-3.0 g/L
Alprazolam (therapeutic) Serum, plasma 10-50 ng/mL 3.24 32-162 nmol/L
Aluminum Serum, plasma <6 ng/mL 37.06 0.0-222.4 nmol/L

Amikacin (therapeutic, peak) Serum, plasma 20-30 μg/mL 1.71 34-52 μmol/L
Amino acid fractionation

Alanineb Plasma 1.87-5.89 mg/dL 112.2 210-661 μmol/L
α-Aminobutyric acidb Plasma 0.08-0.36 mg/dL 97 8-35 μmol/L
Arginineb Plasma 0.37-2.40 mg/dL 57.4 21-138 μmol/L
Asparagineb Plasma 0.40-0.91 mg/dL 75.7 30-69 μmol/L
Aspartic acidb Plasma <0.3 mg/dL 75.1 <25 μmol/L
Citrullineb Plasma 0.2-1.0 mg/dL 57.1 12-55 μmol/L
Cystineb Plasma 0.40-1.40 mg/dL 83.3 33-117 μmol/L
Glutamic acidb Plasma 0.2-2.8 mg/dL 67.97 15-190 μmol/L
Glutamineb Plasma 6.1-10.2 mg/dL 68.42 420-700 μmol/L
Glycineb Plasma 0.9-4.2 mg/dL 133.3 120-560 μmol/L
Histidineb Plasma 0.5-1.7 mg/dL 64.5 32-110 μmol/L
Hydroxyprolineb Plasma <0.55 mg/dL 76.3 <42 μmol/L
Isoleucineb Plasma 0.5-1.3 mg/dL 76.24 40-100 μmol/L
Leucineb Plasma 1.0-2.3 mg/dL 76.3 75-175 μmol/L
Lysineb Plasma 1.2-3.5 mg/dL 68.5 80-240 μmol/L
Methionineb Plasma 0.1-0.6 mg/dL 67.1 6-40 μmol/L
Ornithineb Plasma 0.4-1.4 mg/dL 75.8 30-106 μmol/L
Phenylalanineb Plasma 0.6-1.5 mg/dL 60.5 35-90 μmol/L
Prolineb Plasma 1.2-3.9 mg/dL 86.9 104-340 μmol/L
Serineb Plasma 0.7-2.0 mg/dL 95.2 65-193 μmol/L
Taurineb Plasma 0.3-2.1 mg/dL 80 24-168 μmol/L
Threonineb Plasma 0.9-2.5 mg/dL 84 75-210 μmol/L
Tryptophanb Plasma 0.5-1.5 mg/dL 48.97 25-73 μmol/L
Tyrosineb Plasma 0.4-1.6 mg/dL 55.19 20-90 μmol/L
Valineb Plasma 1.7-3.7 mg/dL 85.5 145-315 μmol/L
Continued next page—

CLINICAL LABORATORY REFERENCE VALUES xix
Conversion
Traditional Factor,
α-Aminobutyric acid b
Plasma 0.08-0.36 mg/dL 97 8-35 μmol/L
Amiodarone (therapeutic) Serum, plasma 0.5-2.5 μg/mL 1.55 0.8-3.9 μmol/L
δ-Aminolevulinic acid Urine 1.0-7.0 mg/24 h 7.626 8-53 μmol/day
Amitriptyline (therapeutic) Serum, plasma 80-250 ng/mL 3.61 289-903 nmol/L
Ammonia (as NH3) b

Plasma 15-50 μg/dL 0.714 11-35 μmol/L
Amobarbital (therapeutic) Serum 1-5 μg/mL 4.42 4-22 μmol/L
Amoxapine (therapeutic) Plasma 200-600 ng/mL 1 200-600 μg/L
Amylase a,b
Serum 27-130 U/L 0.017 0.46-2.21 μKat/L
b
Androstenedione, male Serum 75-205 ng/dL 0.0349 2.6-7.2 nmol/L
b
Androstenedione, female Serum 85-275 ng/dL 0.0349 3.0-9.6 nmol/L
Angiotensin I Plasma <25 pg/mL 1 <25 ng/L
Angiotensin II Plasma 10-60 pg/mL 1 10-60 ng/L
Angiotensin-converting Serum 8-52 U/L 0.017 0.14-0.88 μKat/L

enzyme (ACE)a,b
Anion gap (Na+)−(Cl− + HCO3−) Serum, plasma 8-16 mEq/L 1 8-16 nmol/L
Antidiuretic hormone (ADH, Plasma 1-5 pg/mL 0.926 0.9-4.6 pmol/L

vasopressin) (varies with
osmolality: 285-290 mOsm/kg)
α2-Antiplasmin Plasma 80-130 % 0.01 0.8-1.3 Fraction of 1.0
Antithrombin III Plasma 21-30 mg/dL 10 210-300 mg/L
Antithrombin III activity Plasma 80-130 % 0.01 0.8-1.3 Fraction of 1.0
α1-Antitrypsin Serum 80-200 mg/dL 0.01 0.8-2.0 g/L
Apolipoprotein Ab
Male Serum 80-151 mg/dL 0.01 0.8-1.5 g/L
Female Serum 80-170 mg/dL 0.01 0.8-1.7 g/L
Apolipoprotein Bb
Male Serum, plasma 50-123 mg/dL 0.01 0.5-1.2 g/L
Female Serum, plasma 25-120 mg/dL 0.01 0.25-1.20 g/L
Arginineb Plasma 0.37-2.40 mg/dL 57.4 21-138 μmol/L
Arsenic (As) Whole blood <23 μg/L 0.0133 <0.31 μmol/L
Arsenic (As), chronic poisoning Whole blood 100-500 μg/L 0.0133 1.33-6.65 μmol/L
Arsenic (As), acute poisoning Whole blood 600-9300 μg/L 0.0133 7.9-123.7 μmol/L
Ascorbate, ascorbic acid (see vitamin C)
Asparagineb Plasma 0.40-0.91 mg/dL 75.7 30-69 μmol/L
Aspartate aminotransferase Serum 20-48 U/L 0.017 0.34-0.82 μKat/L

(AST, SGOT)a,b
Aspartic acidb Plasma <0.3 mg/dL 75.1 <25 μmol/L
Atrial natriuretic hormone Plasma 20-77 pg/mL 1 20-77 ng/L
Barbiturates (see individual drugs; pentobarbital, phenobarbital, thiopental)
Basophils (see complete blood count, white blood cell count)
Benzodiazepines (see individual drugs; alprazolam, chlordiazepoxide, diazepam, lorazepam)

xx CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
Beryllium, toxic Urine >20 μg/L 0.111 >2.22 μmol/L
Bicarbonate Plasma 21-28 mEq/L 1 21-28 mmol/L
Bile acids (total) Serum 0.3-2.3 μg/mL 2.448 0.73-5.63 μmol/L
Bilirubin
Totalb Serum 0.3-1.2 mg/dL 17.1 2-18 μmol/L
Direct (conjugated) Serum <0.2 mg/dL 17.1 <3.4 μmol/L
Biotin Whole blood, 200-500 pg/mL 0.0041 0.82-2.05 nmol/L

serum
Bismuth Whole blood 1-12 μg/L 4.785 4.8-57.4 nmol/L
Blood gases
PCO2 Arterial blood 35-45 mm Hg 1 35-45 mm Hg
pH Arterial blood 7.35-7.45 — 1 7.35-7.45 —
PO2 Arterial blood 80-100 mm Hg 1 80-100 mm Hg
Blood urea nitrogen (BUN, see urea nitrogen)
BNP Plasma <100 pg/mL 1 <100 pg/mL
Bupropion (Wellbutrin, Zyban) Serum, plasma 25-100 ng/mL 3.62 91-362 nmol/L
C1 esterase inhibitor Serum 12-30 mg/dL 0.01 0.12-0.30 g/L

C3 complement b
Serum 1200-1500 μg/mL 0.001 1.2-1.5 g/L
C4 complement b
Serum 350-600 μg/mL 0.001 0.35-0.60 g/L
CA125 Serum <35 U/mL 1.0 <35 kU/L
CA19-9 Serum <37 U/mL 1.0 <37 kU/L

CA15-3 Serum <30 U/mL 1.0 <30 kU/L
CA27.29 Serum <37.7 U/mL 1.0 <37.7 kU/L
Cadmium (nonsmoker) Whole blood 0.3-1.2 μg/L 8.897 2.7-10.7 nmol/L
Caffeine (therapeutic, infants) Serum, plasma 8-20 μg/mL 5.15 41-103 μmol/L
Calciferol (see vitamin D)
Calcitonin Serum, plasma <19 pg/mL 1 <19 ng/L
Calcium, ionized Serum 4.60-5.08 mg/dL 0.25 1.15-1.27 mmol/L

Calcium, total Serum 8.2-10.2 mg/dL 0.25 2.05-2.55 mmol/L
Calcium, normal diet Urine <250 mg/24 h 0.025 <6.2 mmol/day
Carbamazepine (therapeutic) Serum, plasma 8-12 μg/mL 4.23 34-51 μmol/L
Carbon dioxide Serum, plasma, 22-28 mEq/L 1 22-28 mmol/L

venous blood
Carboxyhemoglobin (carbon monoxide), as fraction of hemoglobin saturation

Nonsmoker Whole blood <2.0 % 0.01 <0.02 Fraction of 1.0
Toxic Whole blood >20 % 0.01 >0.2 Fraction of 1.0
β-Carotene Serum 10-85 μg/dL 0.0186 0.2-1.6 μmol/L
Catecholamines, total (see norepinephrine)

CEA, nonsmoker Serum <3 ng/mL 1.0 <3 μg/L
CEA, smoker Serum <5 ng/mL 1.0 <5 μg/L

b
Ceruloplasmin Serum 20-40 mg/dL 10 200-400 mg/L
Chloramphenicol (therapeutic) Serum 10-25 μg/mL 3.1 31-77 μmol/L

CLINICAL LABORATORY REFERENCE VALUES xxi
Conversion
Traditional Factor,
Chlordiazepoxide (therapeutic) Serum, plasma 0.7-1.0 μg/mL 3.34 2.3-3.3 μmol/L

Chloride Serum, plasma 96-106 mEq/L 1 96-106 mmol/L
Chloride CSF 118-132 mEq/L 1 118-132 mmol/L
Chlorpromazine (therapeutic, adult) Plasma 50-300 ng/mL 3.14 157-942 nmol/L
Chlorpromazine (therapeutic, child) Plasma 40-80 ng/mL 3.14 126-251 nmol/L
Chlorpropamide (therapeutic) Plasma 75-250 mg/L 3.61 270-900 μmol/L
Cholesterol, high-density Plasma 40-60 mg/dL 0.02586 1.03-1.55 mmol/L
lipoproteins (HDL)
Cholesterol, low-density lipoproteins (LDL)b
Optimal Plasma <100 mg/dL 0.02586 <2.59 mmol/L
Near optimal Plasma 100-129 mg/dL 0.02586 2.59-3.34 mmol/L
Borderline high Plasma 130-159 mg/dL 0.02586 3.37-4.12 mmol/L
High Plasma 160-189 mg/dL 0.02586 4.15-4.90 mmol/L
Very high Plasma >190 mg/dL 0.02586 >4.90 mmol/L
Cholesterol (total), adult
Desirable Serum <200 mg/dL 0.02586 <5.17 mmol/L
Borderline high Serum 200-239 mg/dL 0.02586 5.17-6.18 mmol/L
High Serum >240 mg/dL 0.02586 >6.21 mmol/L
Cholesterol (total), children

Desirable Serum <170 mg/dL 0.02586 4.40 mmol/L
Borderline high Serum 170-199 mg/dL 0.02586 4.40-5.15 mmol/L
High Serum >200 mg/dL 0.02586 >5.18 mmol/L
Chromium Whole blood 0.7-28.0 μg/L 19.2 13.4-538.6 nmol/L
Citrate Serum 1.2-3.0 mg/dL 52.05 60-160 μmol/L
Citrulline b
Plasma 0.4-2.4 mg/dL 57.1 20-135 μmol/L
Clonazepam (therapeutic) Serum 15-60 ng/mL 3.17 48-190 nmol/L
Coagulation factor I (fibrinogen) Plasma 150-400 mg/dL 0.01 1.5-4.0 g/L
Coagulation factor II (prothrombin) Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor V Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor VII Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor VIII Plasma 50-200 % 0.01 0.50-2.00 Fraction of 1.0
Coagulation factor IX Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor X Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor XI Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Coagulation factor XII Plasma 60-140 % 0.01 0.60-1.40 Fraction of 1.0
Cobalt Serum <1.0 μg/L 16.97 <17 nmol/L
Codeine (therapeutic) Serum 10-100 ng/mL 3.34 33-334 nmol/L
Complete blood count (CBC)
Hematocritb
Male Whole blood 41-50 % 0.01 0.41-0.50 Fraction of 1.0
Female Whole blood 35-45 % 0.01 0.35-0.45 Fraction of 1.0
Hemoglobin (mass concentration)b
Male Whole blood 13.5-17.5 g/dL 10 135-175 g/L
Female Whole blood 12.0-15.5 g/dL 10 120-155 g/L

xxii CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
Hemoglobin (substance concentration, Hb [Fe])

Male Whole blood 13.6-17.2 g/dL 0.6206 8.44-10.65 mmol/L
Female Whole blood 12.0-15.0 g/dL 0.6206 7.45-9.30 mmol/L
Mean corpuscular hemoglobin Whole blood 27-33 pg/cell 1 27-33 pg/cell
(MCH), mass concentrationb
MCH, substance concentration, Whole blood 27-33 pg/cell 0.06206 1.70-2.05 fmol
Hb [Fe]
Mean corpuscular hemoglobin Whole blood 33-37 g Hb/dL 10 330-370 g Hb/L
concentration (MCHC), mass
concentration
MCHC, substance concentration, Whole blood 33-37 g Hb/dL 0.6206 20-23 mmol/L
Hb [Fe]
Mean cell volume (MCV)b Whole blood 80-100 μm3 1 80-100 fL
Platelet count Whole blood 150-450 103 μL−1 1 150-450 109 L−1
Red blood cell count
Female Whole blood 3.9-5.5 106 μL−1 1 3.9-5.5 1012 L−1
Male Whole blood 4.6-6.0 106 μL−1 1 4.6-6.0 1012 L−1
Reticulocyte countb Whole blood 25-75 103 μL−1 1 25-75 109 L−1
Reticulocyte countb (fraction) Whole blood 0.5-1.5 % of RBCs 0.01 0.005-0.015 Fraction of RBCs
White blood cell countb Whole blood 4.5-11.0 103 μL−1 1 4.5-11.0 109 L−1
Differential countb (absolute)
Neutrophils Whole blood 1800-7800 μL−1 1 1.8-7.8 109 L−1
Bands Whole blood 0-700 μL−1 1 0.00-0.70 109 L−1
Lymphocytes Whole blood 1000-4800 μL−1 1 1.0-4.8 109 L−1
Monocytes Whole blood 0-800 μL−1 1 0.00-0.80 109 L−1
Eosinophils Whole blood 0-450 μL−1 1 0.00-0.45 109 L−1
Basophils Whole blood 0-200 μL−1 1 0.00-0.20 109 L−1
Differential countb (number fraction)
Neutrophils Whole blood 56 % 0.01 0.56 Fraction of 1.0
Bands Whole blood 3 % 0.01 0.03 Fraction of 1.0
Lymphocytes Whole blood 34 % 0.01 0.34 Fraction of 1.0
Monocytes Whole blood 4 % 0.01 0.04 Fraction of 1.0
Eosinophils Whole blood 2.7 % 0.01 0.027 Fraction of 1.0
Basophils Whole blood 0.3 % 0.01 0.003 Fraction of 1.0
Copperb Serum 70-140 μg/dL 0.1574 11.0-22.0 μmol/L
Coproporphyrin Urine <200 μg/24 h 1.527 <300 nmol/day

b
Corticotropin (08:00) Plasma <120 pg/mL 0.22 <26 pmol/L
b
Cortisol, total
08:00 Plasma 5-25 μg/dL 27.6 138-690 nmol/L
16:00 Plasma 3-16 μg/dL 27.6 83-442 nmol/L
20:00 Plasma <50% of 08:00 μg/dL 1 <50% of 08:00 nmol/L
Cortisol, freeb Urine 30-100 μg/24 h 2.76 80-280 nmol/day
Cotinine (smoker) Plasma 16-145 ng/mL 5.68 91-823 nmol/L

C-peptide Serum 0.5-3.5 ng/mL 0.333 0.17-1.17 nmol/L
Creatine, male Serum 0.2-0.7 mg/dL 76.3 15.3-53.3 μmol/L
Creatine, female Serum 0.3-0.9 mg/dL 76.3 22.9-68.6 μmol/L
Creatine kinase (CK) a

Serum 50-200 U/L 0.017 0.85-3.40 μKat/L
CK-MB fraction Serum <6 % 0.01 <0.06 Fraction of 1.0
Creatinineb Serum, plasma 0.6-1.2 mg/dL 88.4 53-106 μmol/L
Creatinine Urine 1-2 g/24 h 8.84 8.8-17.7 mmol/day
Creatinine clearance, glomerular Serum, urine 75-125 mL/min/ 0.00963 0.72-1.2 mL/s/m2
filtration rate 1.73 m2

CLINICAL LABORATORY REFERENCE VALUES xxiii
Conversion
Traditional Factor,
C-telopeptide
Men Serum, plasma 60-700 pg/mL 1 60-700 pg/mL
Premenopausal women Serum, plasma 40-465 pg/mL 1 40-465 pg/mL
Cyanide (toxic) Whole blood >1.0 μg/mL 38.4 >38.4 μmol/L
Cyanocobalamin (see vitamin B12)
Cyclic adenosine Plasma 4.6-8.6 ng/mL 3.04 14-26 nmol/L
monophosphate (cAMP)
Cyclosporine (toxic) Whole blood >400 ng/mL 0.832 >333 nmol/L
Cystineb
D-dimer Plasma Negative ng/mL 1 Negative ng/mL
(<500) (<500)
Dehydroepiandrosterone (DHEA) Plasma, serum 180-1250 ng/dL 0.0347 6.2-43.3 nmol/L
(unconjugated, male)b
Dehydroepiandrosterone sulfate Plasma, serum 10-619 μg/dL 0.027 0.3-16.7 μmol/L
(DHEA-S) (male)b
Desipramine (therapeutic) Plasma, serum 50-200 ng/mL 3.75 170-700 nmol/L
Diazepam (therapeutic) Plasma, serum 100-1000 ng/mL 0.00351 0.35-3.51 μmol/L
Digoxin (therapeutic) Plasma 0.5-2.0 ng/mL 1.281 0.6-2.6 nmol/L
Disopyramide (therapeutic) Plasma, serum 2.8-7.0 mg/L 2.95 8-21 μmol/L
Doxepin (therapeutic) Plasma, serum 150-250 ng/mL 3.58 540-890 nmol/L
Electrolytes
Chloride Serum, plasma 96-106 mEq/L 1 96-106 mmol/L
Carbon dioxide (CO2) Serum, plasma, 22-28 mEq/L 1 22-28 mmol/L
venous blood
Potassium Plasma 3.5-5.0 mEq/L 1 3.5-5.0 mmol/L
Sodiumb Plasma 136-142 mEq/L 1 136-142 mmol/L
Eosinophils (see complete blood count, white blood cell count)
Epinephrine (supine) Plasma <50 pg/mL 5.46 <273 pmol/L
Epinephrineb Urine <20 μg/24 h 5.46 <109 nmol/day
Erythrocyte count (see complete blood count, red blood cell count)
Erythrocyte sedimentation rate (ESR)b Whole blood 0-20 mm/h 1 0-20 mm/h
Erythropoietin Serum 5-36 mU/mL 1 5-36 IU/L
Estradiol (E2, unconjugated),b female
Follicular phase Serum 20-350 pg/mL 3.69 73-1285 pmol/L
Midcycle peak Serum 150-750 pg/mL 3.69 551-2753 pmol/L
Luteal phase Serum 30-450 pg/mL 3.69 110-1652 pmol/L
Postmenopausal Serum <59 pg/mL 3.69 <218 pmol/L
Estradiol (unconjugated),b male Serum <20 pg/mL 3.67 <184 pmol/L
Estriol (E3, unconjugated), males Serum <2 ng/mL 3.47 <6.9 nmol/L
and nonpregnant females, varies
with length of gestation
Estrogens (total),b female
Follicular phase Serum 60-200 pg/mL 1 60-200 ng/L
Luteal phase Serum 160-400 pg/mL 1 160-400 ng/L
Postmenopausal Serum <130 pg/mL 1 <130 ng/L
Estrogens (total),b male Serum 20-80 pg/mL 1 20-80 ng/L

xxiv CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
b
Estrone (E1), female
Follicular phase Plasma, serum 100-250 pg/mL 3.69 370-925 pmol/L
Luteal phase Plasma, serum 15-200 pg/mL 3.69 55-740 pmol/L
Postmenopausal Plasma, serum 15-55 pg/mL 3.69 55-204 pmol/L
Estrone (E1),b male Plasma, serum 15-65 pg/mL 3.69 55-240 pmol/L
Ethanol (ethyl alcohol), toxic Serum, whole >100 mg/dL 0.2171 >21.7 mmol/L
blood
Ethosuximide Plasma, serum 40-100 μg/mL 7.08 283-708 μmol/L
Ethylene glycol (toxic) Plasma, serum >30 mg/dL 0.1611 >5 mmol/L
Everolimus Whole blood 3-15 ng/mL 1.04 3-16 nmol/L
Fatty acids (nonesterified) Plasma 8-25 mg/dL 0.0354 0.28-0.89 mmol/L
Fecal fat (as stearic acid) Stool 2.0-6.0 g/day 1 2-6 g/day
Felbamate Serum, plasma 30-60 μg/mL 4.20 126-252 μmol/L
Ferritin b
Plasma 15-200 ng/mL 1 15-200 μg/L
α-Fetoprotein b
Serum <10 ng/mL 1 <10 μg/L
Fibrinogen Plasma 150-400 mg/dL 0.01 1.5-4.0 g/L
Fibrin breakdown products (fibrin Serum <10 μg/mL 1 <10 mg/L

split products)
Folate (folic acid) Red blood cells 166-640 ng/mL 2.266 376-1450 nmol/L
Folate (folic acid) Serum 5-25 ng/mL 2.266 11-57 nmol/L

b
Follicle-stimulating hormone (FSH, follitropin), female
Follicular phase Serum 1.37-9.9 mIU/mL 1 1.3-9.9 IU/L
Ovulatory phase Serum 6.17-17.2 mIU/mL 1 6.1-17.2 IU/L
Luteal phase Serum 1.09-9.2 mIU/mL 1 1.0-9.2 IU/L
Postmenopausal Serum 19.3-100.6 mIU/mL 1 19.3-100.6 IU/L
FSH (follitropin),b male Serum 1.42-15.4 mIU/mL 1 1.4-15.4 IU/L

b
FSH (follitropin), female Urine 2-15 IU/24 h 1 2-15 IU/day
b
FSH (follitropin), male Urine 3-12 IU/24 h 1 3-11 IU/day
b
Fructosamine Serum 1.5-2.7 mmol/L 1 1.5-2.7 mmol/L
Gabapentin Serum, plasma 2-20 μg/mL 5.84 12-117 μmol/L

Gastrin (fasting) Serum <100 pg/mL 1 <100 ng/L
Gentamicin (therapeutic, peak) Serum 6-10 μg/mL 2.1 12-21 μmol/L

b
Glucagon Plasma 20-100 pg/mL 1 20-100 ng/L
b
Glucose Serum, plasma 70-110 mg/dL 0.05551 3.9-6.1 mmol/L
Glucose CSF 50-80 mg/dL 0.05551 2.8-4.4 mmol/L
Glucose-6-phosphate Red blood cells 10-14 U/g of Hb 0.0645 0.65-0.90 U/mol of Hb

dehydrogenase
Glutamic acidb Plasma 0.2-2.8 mg/dL 67.97 15-190 μmol/L
Glutamine Plasma 6.1-10.2 mg/dL 68.42 420-700 μmol/L
γ-Glutamyltransferase (GGT; γ-glutamyl transpeptidase) b
Female Serum <30 U/L 0.017 0.51 μKat/L
Male Serum <50 U/L 0.017 <0.85 μKat/L

CLINICAL LABORATORY REFERENCE VALUES xxv
Conversion
Traditional Factor,
Glycerol (free) b
Serum <1.5 mg/dL 0.1086 <0.16 mmol/L
Glycine b
Glycated hemoglobin (hemoglobin A1, A1c)
Whole blood Whole blood 4-5.6 % of total Hb 1 4-5.6 Fraction of

total Hb
Diagnosis Whole blood >6.5 % of total Hb 1 >6.5 Fraction of

total Hb
Gold (therapeutic) Serum 100-200 μg/dL 0.05077 5.1-10.2 μmol/L
Growth hormone, adult (GH, Plasma, serum <10 ng/mL 1 <10 μg/L
somatotropin)b
Haloperidol (therapeutic) Serum, plasma 5-20 ng/mL 2.6 13-52 nmol/L
b
Haptoglobin Serum 40-180 mg/dL 0.01 0.4-1.8 g/L
Hematocrit (see complete blood count)
Hemoglobin (see complete blood count)
Hemoglobin A1c (see glycated hemoglobin)
Hemoglobin A2b Whole blood 2.0-3.5 % total Hb 2.0-3.5 Fraction of 1.0
b
Hemoglobin F (fetal hemoglobin Whole blood <2 % 0.01 <2 Fraction of 1.0
in adult)
Histidineb Plasma 0.5-1.7 mg/dL 64.5 32-110 μmol/L
Homocysteine (total) Plasma, serum 4-12 μmol/L 1 4-12 μmol/L
Homovanillic acid b
Urine <8 mg/24 h 5.489 <45 μmol/day
Human chorionic gonadotropin Serum <3 mIU/mL 1 <3 IU/L
(hCG) (nonpregnant adult female)
β-Hydroxybutyric acid Serum 0.21-2.81 mg/dL 96.05 20-270 μmol/L
5-Hydroxyindoleacetic acid (5-HIAA) Urine <25 mg/24 h 5.23 <131 μmol/day
b
17α-Hydroxyprogesterone, female
Follicular phase Serum 15-70 ng/dL 0.03 0.4-2.1 nmol/L
Luteal phase Serum 35-290 ng/dL 0.03 1.0-8.7 nmol/L
Postmenopausal Serum <70 ng/dL 0.03 <2.1 nmol/L
17α-Hydroxyprogesterone,b male Serum 27-199 ng/dL 0.03 0.8-6.0 nmol/L
Hydroxyproline Plasma <0.55 mg/dL 76.3 <42 μmol/L
5-Hydroxytryptamine (see serotonin)
Ibuprofen (therapeutic) Serum, plasma 10-50 μg/mL 4.85 49-243 μmol/L
Imipramine (therapeutic) Serum, plasma 150-250 ng/mL 3.57 536-893 nmol/L
b
Immunoglobulin A (IgA) Serum 50-350 mg/dL 0.01 0.5-3.5 g/L
Immunoglobulin D (IgD) Serum 0.5-3.0 mg/dL 10 5-30 mg/L
Immunoglobulin E (IgE) Serum 10-179 IU/mL 2.4 24-430 μg/L
b
Immunoglobulin G (IgG) Serum 600-1560 mg/dL 0.01 6.0-15.6 g/L
Immunoglobulin M (IgM)b Serum 54-222 mg/dL 0.01 0.5-2.2 g/L
Insulin Plasma 5-20 μU/mL 6.945 34.7-138.9 pmol/L
Inhibin A
Males Serum 1.0-3.6 pg/mL 1 1.0-3.6 ng/L

xxvi CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
Female, early follicular Serum 5.5-28.2 pg/mL 1 5.5-28.2 ng/L
Female, late follicular Serum 19.5-102.3 pg/mL 1 19.5-102.3 ng/L
Female, midcycle Serum 49.9-155.5 pg/mL 1 49.9-155.5 ng/L
Female, midluteal Serum 13.2-159.6 pg/mL 1 13.2-159.6 ng/L
Female, postmenopausal Serum 1.0-3.9 pg/mL 1 1.0-3.9 ng/L
Insulin C-peptide (see C-peptide)

Insulin-like growth factorb Serum 130-450 ng/mL 1 130-450 μg/L
Ionized calcium (see calcium)
Iron (total)b Serum 60-150 μg/dL 0.179 10.7-26.9 μmol/L
Iron-binding capacity Serum 250-400 μg/dL 0.179 44.8-71.6 μmol/L
Isoleucine b
Isoniazid (therapeutic) Plasma or 1-7 μg/mL 7.29 7-51 μmol/L

serum
Isopropanol (toxic) Plasma, serum >400 mg/L 0.0166 >6.64 mmol/L
Lactate (lactic acid) Arterial blood 3-11.3 mg/dL 0.111 0.3-1.3 mmol/L
Lactate (lactic acid) Venous blood 4.5-19.8 mg/dL 0.111 0.5-2.2 mmol/L
Lactate dehydrogenase (LDH) Serum 50-200 U/L 1 50-200 U/L
Lamotrigine Serum, plasma 2.5-15 μg/dL 3.91 10-59 μmol/L
Lead Whole blood <25 μg/dL 0.0483 <1.21 μmol/L
Leucine b
Leukocyte count (see complete blood count, white blood cell count)
Levetiracetam Serum, plasma 12-46 μg/mL 5.88 71-270 μmol/L

Lidocaine (therapeutic) Serum, plasma 1.5-6.0 μmL g/mL 4.27 6.4-25.6 μmol/L
Lipase a
Serum 0-160 U/L 0.017 0-2.72 μKat/L
Lipoprotein(a) (Lp(a)) Serum, plasma 10-30 mg/dL 0.01 0.1-0.3 g/L
Lithium (therapeutic) Serum, plasma 0.6-1.2 mEq/L 1 0.6-1.2 mmol/L
Lorazepam (therapeutic) Serum, plasma 50-240 ng/mL 3.11 156-746 nmol/L

b
LDL cholesterol Serum, plasma 60-130 mg/dL 0.02586 1.55-3.37 mmol/L
b
Luteinizing hormone (LH), female
Follicular phase Serum 2.0-15.0 mIU/L 1 2.0-15.0 IU/L
Ovulatory peak Serum 22.0-105.0 mIU/L 1 22.0-105.0 IU/L
Luteal phase Serum 0.6-19.0 mIU/L 1 0.6-19.0 IU/L
Postmenopausal Serum 16.0-64.0 mIU/L 1 16.0-64.0 IU/L
LH,b male Serum 2.0-12.0 mIU/L 1 2.0-12.0 IU/L
Lymphocytes (see complete blood count, white blood cell count)
Lysineb Plasma 1.2-3.5 mg/dL 68.5 80-240 μmol/L
Lysozyme (muramidase) Serum 4-13 mg/L 1 4-13 mg/L
Magnesiumb Serum 1.5-2.5 mg/dL 0.4114 0.62-1.03 mmol/L
Magnesiumb Serum 1.3-2.1 mEq/L 0.5 0.65-1.05 mmol/L

Manganese Whole blood 10-12 μg/L 18.2 182-218 nmol/L

CLINICAL LABORATORY REFERENCE VALUES xxvii
Conversion
Traditional Factor,
Maprotiline (therapeutic) Plasma, serum 200-600 ng/mL 1 200-600 μg/L
MCH (see complete blood count)
MCHC (see complete blood count)
Meperidine (therapeutic) Serum, plasma 0.4-0.7 μg/mL 4.04 1.6-2.8 μmol/L
Mercury Whole blood 0.6-59.0 μg/L 4.99 3.0-294.4 nmol/L
Metanephrines (total) b
Urine <1.0 mg/24 h 5.07 <5 μmol/day
Methadone (therapeutic) Serum, plasma 100-400 ng/mL 0.00323 0.32-1.29 μmol/L
Methanol Whole blood, <1.5 mg/L 0.0312 <0.05 mmol/L

serum
Methemoglobin Whole blood <0.24 g/dL 155 <37.2 μmol/L
Methemoglobin Whole blood <1.0 % of total Hb 0.01 <0.01 Fraction of

total Hb
Methionineb Plasma 0.1-0.6 mg/dL 67.1 6-40 μmol/L
Methsuximide (therapeutic) Serum, plasma 10-40 μg/mL 5.29 53-212 μmol/L
Methyldopa (therapeutic) Serum, plasma 1-5 μg/mL 4.73 5-24 μmol/L
Metoprolol (therapeutic) Serum, plasma 75-200 ng/mL 3.74 281-748 nmol/L

Mexthotrexate
Toxic 24 h after dose Serum, plasma ≥10 μmol/L 1 ≥10 μmol/L
Toxic 48 h after dose Serum, plasma ≥1 μmol/L 1 ≥1 μmol/L
Toxic 72 h after dose Serum, plasma ≥0.1 μmol/L 1 ≥0.1 μmol/L

β2-Microglobulin Serum <2 μg/mL 85 <170 nmol/L
Monocytes (see complete blood count, white blood cell count)
Morphine (therapeutic) Serum, plasma 10-80 ng/mL 3.5 35-280 nmol/L

Muramidase (see lysozyme)
Mycophenolic acid Serum, plasma 1.3-3.5 μg/mL 3.12 4-11 μmol/L
Naproxen (therapeutic trough) Plasma, serum >50 μg/mL 4.34 >217 μmol/L
Neutrophils (see complete blood count, white blood cell count)
Niacin (nicotinic acid) Urine 2.4-6.4 mg/24 h 7.3 17.5-46.7 μmol/day
Nickel Whole blood 1.0-28.0 μg/L 17 17-476 nmol/L
Nicotine (smoker) Plasma 0.01-0.05 mg/L 6.16 0.062-0.308 μmol/L
Norepinephrineb Plasma 110-410 pg/mL 5.91 650-2423 nmol/L
Norepinephrineb Urine 15-80 μg/24 h 5.91 89-473 nmol/day
Nortriptyline (therapeutic) Serum, plasma 50-150 ng/mL 3.8 190-570 nmol/L
N-telopeptide (BCE, bone collagen equivalents)
Men Serum 5.4-24.2 nmol BCE/L 1 5.4-24.2 nmol BCE/L

Premenopausal women Serum 6.2-19.0 nmol BCE/L 1 6.2-19.0 nmol BCE/L
Ornithineb Plasma 0.4-1.4 mg/dL 75.8 30-106 μmol/L
Osmolalityb Serum 275-295 mOsm/kg H2O 1 275-295 mmol/kg H2O
Osmolality Urine 250-900 mOsm/kg H2O 1 250-900 mmol/kg H2O

xxviii CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
Osteocalcin b
Serum 3.0-13.0 ng/mL 1 3.0-13.0 μg/L
Oxalate Serum 1.0-2.4 mg/L 11.4 11-27 μmol/L
Oxazepam (therapeutic) Serum, plasma 0.2-1.4 μg/mL 3.49 0.7-54.9 μmol/L
Oxycodone (therapeutic) Plasma, serum 10-100 ng/mL 3.17 32-317 nmol/L
Oxygen, partial pressure (PO2) Arterial blood 80-100 mm Hg 1 80-100 mm Hg
Pantothenic acid (see vitamin B5)
Parathyroid hormone
Intactb Serum 10-50 pg/mL 1 10-50 ng/L
N-terminal specificb Serum 8-24 pg/mL 1 8-24 ng/L
C-terminal (mid-molecule) Serum 0-340 pg/mL 1 0-340 ng/L
Pentobarbital (therapeutic) Serum, plasma 1-5 μg/mL 4.42 4.0-22 μmol/L
Pepsinogen Ib Serum 28-100 ng/mL 1 28-100 μg/L
pH (see blood gases)
Phenobarbital (therapeutic) Serum, plasma 15-40 μg/mL 4.31 65-172 μmol/L

Phenylalanineb Plasma 0.6-1.5 mg/dL 60.5 35-90 μmol/L
Phenytoin (therapeutic) Serum, plasma 10-20 μg/mL 3.96 40-79 μmol/L
Phosphatase, tartrate-resistant acid Serum 1.5-4.5 U/L 0.017 0.03-0.08 μKat/L
Phosphorus (inorganic)b Serum 2.3-4.7 mg/dL 0.3229 0.74-1.52 mmol/L
Phosphorus (inorganic)b Urine 0.4-1.3 g/24 h 32.29 12.9-42.0 mmol/day
Plasminogen Plasma 8.4-14.0 mg/dL 10 84-140 mg/L
Plasminogen Plasma 80-120 % 0.01 0.80-1.20 Fraction of 1.0
Plasminogen activator inhibitor Plasma <15 IU/mL 1 <15 kIU/L

Platelet count (see complete blood count, platelet count)
Porphobilinogen deaminase Red blood cells >7.0 nmol/s/L 1 >7.0 nmol/(s L)
Potassium Plasma 3.5-5.0 mEq/L 1 3.5-5.0 mmol/L
Prealbumin—transthyretin Serum, plasma 18-45 mg/dL 0.01 0.18-0.45 g/L

Pregnanediol,b female
Follicular phase Urine <2.6 mg/24 h 3.12 <8 μmol/day
Luteal phase Urine 2.3-10.6 mg/24 h 3.12 8-33 μmol/day
Pregnanediol,b male Urine 0-1.9 mg/24 h 3.12 0-5.9 μmol/day
Pregnanetriolb Urine <2.5 mg/24 h 2.97 <7.5 μmol/day
Primidone (therapeutic) Serum, plasma 5-12 μg/mL 4.58 23-55 μmol/L
Procainamide (therapeutic) Serum, plasma 4-10 μg/mL 4.23 17-42 μmol/L
Progesterone,b female
Follicular phase Serum 0.1-0.7 ng/mL 3.18 0.5-2.2 nmol/L
Luteal phase Serum 2.0-25.0 ng/mL 3.18 6.4-79.5 nmol/L
Progesterone,b male Serum 0.13-0.97 ng/mL 3.18 0.4-3.1 nmol/L
Prolactin (nonlactating subject) Serum 1-25 ng/mL 1 1-25 μg/L
Proline b
Propoxyphene (therapeutic) Serum 0.1-0.4 μg/mL 2.946 0.3-1.2 μmol/L
Propanolol (therapeutic) Serum, plasma 50-100 ng/mL 3.86 190-386 nmol/L

CLINICAL LABORATORY REFERENCE VALUES xxix
Conversion
Traditional Factor,
b
Protein (total) Serum 6.0-8.0 g/dL 10 60-80 g/L
Protein C Plasma 70-140 % 0.01 0.70-1.40 Fraction of 1.0
Protein electrophoresis (serum protein electrophoresis [SPEP]), fraction of total protein

Albumin Serum 52-65 % 0.01 0.52-0.65 Fraction of 1.0
α1-Globulin Serum 2.5-5.0 % 0.01 0.025-0.05 Fraction of 1.0
α2-Globulin Serum 7.0-13.0 % 0.01 0.07-0.13 Fraction of 1.0
β-Globulin Serum 8.0-14.0 % 0.01 0.08-0.14 Fraction of 1.0
γ-Globulin Serum 12.0-22.0 % 0.01 0.12-0.22 Fraction of 1.0
Protein electrophoresis (SPEP), concentration

Albumin Serum 3.2-5.6 g/dL 10 32-56 g/L
α1-Globulin Serum 0.1-0.4 g/dL 10 1-10 g/L
α2-Globulin Serum 0.4-1.2 g/dL 10 4-12 g/L
β-Globulin Serum 0.5-1.1 g/dL 10 5-11 g/L
γ-Globulin Serum 0.5-1.6 g/dL 10 5-16 g/L
Protein S (activity) Plasma 70-140 % 0.01 0.70-1.40 Fraction of 1.0
Prothrombin time (PT) Plasma 10-13 s 1 10-13 s
Protoporphyrin Red blood cells 15-50 μg/dL 0.0177 0.27-0.89 μmol/L

PSA Serum 0-4.0 ng/mL 1 0-4.0 μg/L
Pyridinium cross-links (deoxypyridinoline)
Male Urine 10.3-20 nmol/mmol 1 10.3-20 nmol/mmol
creatinine creatinine
Premenopausal female Urine 15.3-33.6 nmol/mmol 1 15.3-33.6 nmol/mmol
creatinine creatinine
Pyridoxine (see vitamin B6)
Pyruvate (as pyruvic acid) Whole blood 0.3-0.9 mg/dL 113.6 34-102 μmol/L
Quinidine (therapeutic) Serum, plasma 2.0-5.0 μg/mL 3.08 6.2-15.4 μmol/L
Red blood cell count (see complete blood count)
Red cell folate (see folate)
Renin (normal-sodium diet)b Plasma 1.1-4.1 ng/mL/h 1 1.1-4.1 ng/(mL h)
Reticulocyte count b
Whole blood 25-75 10 μL
3 −1
1 25-75 109 L−1
Reticulocyte countb (fraction) Whole blood 0.5-1.5 % of RBCs 0.01 0.005-0.015 Fraction of RBCs
Retinol (see vitamin A)
Rheumatoid factor Serum <30 IU/mL 1 <30 kIU/L
Riboflavin (see vitamin B2)
Salicylates (therapeutic) Serum, plasma 15-30 mg/dL 0.0724 1.08-2.17 mmol/L
Sedimentation rate (see erythrocyte sedimentation rate)
Selenium Whole blood 58-234 μg/L 0.0127 0.74-2.97 μmol/L
Serineb Plasma 0.7-2.0 mg/dL 95.2 65-193 μmol/L
Serotonin (5-hydroxytryptamine) Whole blood 50-200 ng/mL 0.00568 0.28-1.14 μmol/L
Sertraline (Zoloft) Serum or plasma 10-50 ng/mL 3.27 33-164 nmol/L
SPEP (see protein electrophoresis)
Sex hormone-binding globulinb Serum 0.5-1.5 μg/dL 34.7 17.4-52.1 nmol/L
Sirolimus Whole blood 4-20 ng/mL 1.1 4-22 nmol/L

xxx CLINICAL LABORATORY REFERENCE VALUES
Conversion
Traditional Factor,
b
Sodium Plasma 136-142 mEq/L 1 136-142 mmol/L
Somatostatin Plasma <25 pg/mL 1 <25 ng/L
Somatomedin C (see insulin-like growth factor)
Strychnine (toxic) Whole blood >0.5 mg/L 2.99 >1.5 μmol/L
Substance P Plasma <240 pg/mL 1 <240 ng/L
Sulfhemoglobin Whole blood <1.0 % of total Hb 0.01 <0.010 Fraction of

total Hb
Tacrolimus Whole blood 3-20 ng/mL 1.24 4-25 nmol/L
Taurine b
b
Testosterone, male Plasma, serum 300-1200 ng/dL 0.0347 10.4-41.6 nmol/L
Testosterone, female b
Plasma, serum <85 ng/dL 0.0347 2.95 nmol/L
Theophylline (therapeutic) Plasma, serum 10-20 μg/mL 5.55 56-111 μmol/L
Thiamine (see vitamin B1)
Thiocyanate (nonsmoker) Plasma, serum 1-4 mg/L 17.2 17-69 μmol/L
Thiopental (therapeutic) Plasma, serum 1-5 μg/mL 4.13 4-21 μmol/L
Thioridazine (therapeutic) Plasma, serum 1.0-1.5 μg/mL 2.7 2.7-4.1 μmol/L

Thrombin time Plasma 16-24 s 1.0 16-24 s
Threonine b
Thyroglobulin b
Serum 3-42 ng/mL 1 3-42 μg/L
Thyrotropin (thyroid-stimulating Serum 0.5-5.0 μIU/mL 1 0.5-5.0 mU/L

hormone, TSH)b
Thyroxine, free (FT4)b Serum 0.9-2.3 ng/dL 12.87 12-30 pmol/L
Thyroxine, total (T4)b Serum 5.5-12.5 μg/dL 12.87 71-160 nmol/L
Thyroxine-binding globulin (TBG),b Serum 10-26 μg/dL 12.9 129-335 nmol/L

as T4 binding capacity
Tissue plasminogen activator Plasma <0.04 IU/mL 1000 <40 IU/L

Tobramycin (therapeutic, peak) Plasma, serum 5-10 μg/mL 2.14 10-21 μmol/L
Tocainide (therapeutic) Plasma, serum 4-10 μg/mL 5.2 21-52 μmol/L
α-Tocopherol (see vitamin E)
Topiramate Serum, plasma 5-20 μg/mL 2.95 15-59 μmol/L

b
Transferrin (siderophilin) Serum 200-380 mg/dL 0.01 2.0-3.8 g/L
b
Triglycerides Plasma, serum 10-190 mg/dL 0.01129 0.11-2.15 mmol/L
b
Triiodothyronine, free (FT3) Serum 260-480 pg/dL 0.0154 4.0-7.4 pmol/L
b
Triiodothyronine, resin uptake Serum 25-35 % 0.01 0.25-0.35 Fraction of 1.0
b
Triiodothyronine, total (T3) Serum 70-200 ng/dL 0.0154 1.08-3.14 nmol/L
Troponin I (cardiac) Serum 0-0.4 ng/mL 1 0-0.4 μg/L
Troponin T (cardiac) Serum 0-0.1 ng/mL 1 0-0.1 μg/L
Tryptophan b
Tyrosine b
b
Urea nitrogen (BUN) Serum 8-23 mg/dL 0.0357 2.9-8.2 mmol/L

CLINICAL LABORATORY REFERENCE VALUES xxxi
Conversion
Traditional Factor,
b
Uric acid Serum 4.0-8.5 mg/dL 0.0595 0.24-0.51 mmol/L
Urobilinogen b
Urine 0.05-2.5 mg/24 h 1.693 0.1-4.2 μmol/day
Valine b
Valproic acid (therapeutic) Plasma, serum 50-150 μg/mL 6.93 346-1040 μmol/L
Vancomycin (therapeutic, peak) Plasma, serum 10-20 μg/mL 0.69 6.9-13.8 μmol/L
Vanillylmandelic acid (VMA) b

Urine 2.1-7.6 mg/24 h 5.046 11-38 μmol/day
Vasoactive intestinal polypeptide Plasma <50 pg/mL 1 <50 ng/L
Verapamil (therapeutic) Plasma, serum 100-500 ng/mL 2.2 220-1100 nmol/L
Vitamin A (retinol) b
Serum 30-80 μg/dL 0.0349 1.05-2.80 μmol/L
Vitamin B1 (thiamine) Whole blood 2.5-7.5 μg/dL 29.6 74-222 nmol/L
Vitamin B2 (riboflavin) Plasma, serum 4-24 μg/dL 26.6 106-638 nmol/L
Vitamin B5 (pantothenic acid) Whole blood 0.2-1.8 μg/mL 4.56 0.9-8.2 μmol/L
Vitamin B6 (pyridoxine) Plasma 5-30 ng/mL 4.046 20-121 nmol/L

b
Vitamin B12 (cyanocobalamin) Serum 160-950 pg/mL 0.7378 118-701 pmol/L
Vitamin C (ascorbic acid) Plasma, serum 0.4-1.5 mg/dL 56.78 23-85 μmol/L
Vitamin D, 1,25-dihydroxyvitamin D Plasma, serum 16-65 pg/mL 2.6 42-169 pmol/L
Vitamin D, 25-hydroxyvitamin D Plasma, serum 14-60 ng/mL 2.496 35-150 nmol/L

Vitamin E (α-tocopherol) b
Plasma, serum 0.5-1.8 mg/dL 23.22 12-42 μmol/L
Vitamin K Plasma, serum 0.13-1.19 ng/mL 2.22 0.29-2.64 nmol/L
von Willebrand factor (ranges vary Plasma 70-140 % 0.01 0.70-1.40 Fraction of 1.0
according to blood type)
Warfarin (therapeutic) Plasma, serum 1.0-10 μg/mL 3.24 3.2-32.4 μmol/L
White blood cell count b

Whole blood 4.5-11.0 10 μL
3 −1
1 4.5-11.0 109 L−1
White blood cell, differential count (see complete blood count)
Xylose absorption test (25-g dose)b Whole blood >25 mg/dL mg/dL 0.06661 >1.7 mmol/L
Zidovudine (therapeutic) Plasma, serum 0.15-0.27 μg/mL 3.74 0.56-1.01 μmol/L
Zinc Serum 50-150 μg/dL 0.153 7.7-23.0 μmol/L
The sample type listed under Specimen in this table shows the reference interval for that specimen type. Thus, if the specimen for a test is listed as serum, the reference interval
shown is for serum specimens. For many tests listed with serum as the specimen type, plasma is also acceptable, often with a similar reference interval.
The normal ranges listed here are included as a helpful guide and are by no means comprehensive. The listed reference, unless noted, pertains to adults. Laboratory results
are method dependent and can have intralaboratory variation. Conversion factors are not affected by age-related differences. This table is compiled from data in the following
sources: 1) Tietz NW, ed. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia: WB Saunders Co; 1995; 2) Laposata M. SI Unit Conversion Guide. Boston: NEJM Books; 1992;
3) American Medical Association Manual of Style: A Guide for Authors and Editors. 9th ed. Chicago: AMA; 1998:486–503. Copyright 1998, American Medical Association;
4) Jacobs DS, DeMott WR, Oxley DK, eds. Jacobs & DeMott Laboratory Test Handbook With Key Word Index. 5th ed. Hudson, OH: Lexi-Comp Inc; 2001; 5) Henry JB, ed. Clinical
Diagnosis and Management by Laboratory Methods. 20th ed. Philadelphia: WB Saunders Co; 2001; 6) Kratz A, et al. Laboratory reference values. N Engl J Med. 2006;351:1548–1563;
7) Burtis CA, ed. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed. St. Louis: Elsevier; 2012. This version of the table of reference ranges was reviewed and
updated by Jessica Franco-Colon, PhD, and Kay Brooks.
a
The SI unit katal is the amount of enzyme generating 1 mol of product per second. Although provisionally recommended as the SI unit for enzymatic activity, it has not been
universally accepted. It is suitable to maintain use of U/L in these circumstances (conversion factor 1.0).
b
For this analyte, there is age dependence for the reference range. There may be several different normal ranges for different pediatric age ranges. Consult your clinical
laboratory for the local institution age-specific reference range. Pediatric reference values may also be found in Soldin SJ, Brugnara C, Wong EC, eds; Hicks JM, editor emeritus.
Pediatric References Intervals. 5th ed. (formerly Pediatric Reference Ranges). Washington, DC: AACC Press; 2005.
C H A P T E R
Concepts in
Laboratory Medicine
Michael Laposata 1
LEARNING OBJECTIVES
1. To understand the concepts of sensitivity, specificity, predictive value,
prevalence, and incidence.
2. To learn the frequently encountered preanalytical variables that influence
laboratory test results.
3. To identify the well-known interferences in many of the laboratory tests.
4. To understand the individual steps in specimen processing and handling.
5. To understand the guidelines for appropriate selection of laboratory tests.
6. To understand how cell injury and inflammation result in the generation
of plasma markers of these processes.
CHAPTER OUTLINE
Analytical and Statistical Concepts Preanalytical Variables that Affect Laboratory
in Data Analysis 2 Test Results 7
Ranges Used in the Interpretation The Effect of Age on Laboratory Tests 7
of Test Results 2 The Effect of Gender on Laboratory Tests 7
The Reference Range 2 The Effect of Body Mass on Laboratory Tests 8
The Desirable Range 2 Preparation of the Patient for
Therapeutic Range 2 Laboratory Testing 8
Interpretations of Clinical Laboratory Patient Posture for Blood Collection 8
Test Results that Do Not Involve the Differences in Test Results Between Samples
Use of a Range 3 of Venous, Arterial, and Capillary Blood 8
The Need for a Diagnostic Cutoff 3 Interferences in Laboratory Tests 8
The Definition of Sensitivity of Analytical Interferences in Laboratory Testing 8
a Laboratory Test 4 Impact of Drugs on Laboratory Test Results 8
The Definition of Specificity of Test Selection Guidelines 9
a Laboratory Test 4 The Use of Screening Tests Before
The Identification of the Appropriate Value Esoteric Tests 9
for the Diagnostic Threshold 4 The Danger of Ordering Too Many
The Definition of Predictive Value of Laboratory Tests 9
a Positive Test 5 Specimen Processing and Handling 9
The Definition of Predictive Value of The Importance of Turnaround Time 9
a Negative Test 5 Tubes for Blood Collection 9
The Difference Between Prevalence Timing of Blood Collection 9
and Incidence 6 Effects of Cell Injury and Inflammation
Precision versus Accuracy 6 on Selected Laboratory Tests 10
Analyzing Errors in Laboratory The Release of Plasma Markers of Organ
Performance 6 Damage from Injured Cells 10
Minimizing Errors in the Selection Markers of Inflammation and the
of Laboratory Tests 7 Acute-phase Response 10
Minimizing Errors in the Interpretation The Serologic Diagnosis of Infectious
of Laboratory Test Results 7 Disease 11
1
2 CHAPTER 1 Concepts in Laboratory Medicine
A
n understanding of the principles set forth in this chapter is essential for the
appropriate selection of laboratory tests and the accurate interpretation of the test
results.
ANALYTICAL AND STATISTICAL CONCEPTS

IN DATA ANALYSIS
Ranges Used in the Interpretation of Test Results
In clinical practice, the laboratory test result is typically placed alongside a range of values for that
test. In most cases, this is the reference range, which is often considered to be the normal range.
It is important to understand that individuals with values inside the reference range may have
subclinical disease, despite the presence of an apparently normal value. The reference range is
dependent on the instrument and reagent used to perform the test. The reference ranges are ide-
ally established inside the laboratory where the test is being performed. Reference ranges supplied
by instrument and reagent manufacturers are not likely to correspond perfectly to ranges gener-
ated within an individual laboratory. This is because the population used to establish the range by
the manufacturer and/or the instruments and reagents used by the manufacturer are likely to be
different from those in an individual clinical laboratory.
The Reference Range

To obtain a reference range, individuals without disease and on no medications donate samples
for testing. A distribution of these values, which should be numerous enough to be statistically
reliable, is plotted. The data are not always distributed in a Gaussian pattern. Therefore, statisti-
To obtain a reference cal methods that are nonparametric are used to identify the central 95% of values. This range,
range, individuals representing the middle 95% of results, is the reference range. As an indication that being outside
without disease and on the reference range does not always reflect the disease, 5% of normal healthy, nonmedicated indi-
no medications donate viduals who donated samples for the reference range determination now fall outside of what has
samples for testing. The become the reference range for the test.
middle 95% of results is
the reference range.
The Desirable Range
Several decades ago, the results for cholesterol testing demonstrated that individuals eating a
high-fat diet showed high cholesterol levels that were associated with atherosclerotic vascular dis-
ease. When these apparently healthy, nonmedicated individuals provided samples for reference
range determinations, the central 95% of values from this population provided an inappropriately
high reference range. Therefore, the use of the classical reference range for selected laboratory
tests in certain populations was not recommended. For that reason, desirable or prognosis-related
ranges were developed. These are commonly established by groups of experts associating labora-
tory test results with clinical outcome.
Therapeutic Range
For certain medications, a therapeutic window exists to provide a target for a blood, plasma,
or serum level for the medication. Values below the therapeutic range typically reflect an inad-
equate amount of medication, and values above the therapeutic range may be associated with a
particular toxic effect. In some cases, the therapeutic range does not reflect the amount of medi-
cation in the blood, but instead reflects a therapeutic effect produced by the drug. For example,
patients taking the drug warfarin are not monitored with warfarin levels in the blood. Instead,
the warfarin decreases the level of coagulation factors, which results in a prolonged prothrom-
bin time (PT), and a calculated value known as the international normalized ratio (INR). The
therapeutic range of warfarin, therefore, is determined by its effect rather than its concentration
in the blood.
CHAPTER 1 Concepts in Laboratory Medicine 3
10 patients with disease &

10 patients without disease
Low value = no disease High value = disease

Test results
Diagnostic threshold
completely separates
positive and negative
test results
No disease Disease
FIGURE 1–1 A clinical situation in which the diagnostic threshold completely separates those with
disease from those without disease.
Interpretations of Clinical Laboratory Test Results

that Do Not Involve the Use of a Range
For certain laboratory tests, the presence of disease is associated with a value that is above a For certain laboratory
threshold. The use of troponin as a marker for myocardial infarction involves a threshold value, tests, the presence of
such that a level above the threshold is consistent with cardiac ischemia. Another prominent disease is associated with
example is related to the detection of drugs of abuse. Any level above zero, as a threshold value, a value that is above a
threshold.
provides evidence for the ingestion of an illicit drug.
For laboratory tests that show too much variability to permit the use of a range or a threshold,
an individual laboratory result for a specific patient can be compared with a result for that same
patient that was generated previously. The longitudinal analysis of results over time can indicate
the progression or regression of the disease.
The Need for a Diagnostic Cutoff

Figure 1–1 shows 2 populations of individuals and their results for a particular test. All of the indi-
viduals who do not have disease have a low value for the test, and all of the individuals with disease
have a high value for the test. There is no overlap between groups in Figure 1–1. In Figure 1–2,
a more commonly encountered situation is shown. There is overlap in laboratory values between
individuals with disease and those without disease. This means that the diagnostic threshold will
necessarily misclassify some patients to create false-positives, false-negatives, or both.
cannot separate
test results
Test results
Line drawn to
maximize sensitivity—
False-
identifies all those
positives
with disease correctly
No disease Disease
FIGURE 1–2 A clinical situation in which a diagnostic threshold is selected to maximize sensitivity.
The Definition of Sensitivity of a Laboratory Test

The population of individuals who have disease is the focus of sensitivity. The sensitivity of a labo-
ratory test is its capacity to identify all individuals with disease. The threshold used in Figure 1–2
maximizes sensitivity by placing all those with disease above the line. This placement of the diag-
nostic threshold would decrease the number of false-negatives (those with disease who fall below
the line), because everybody with the disease would have a positive test result. However, there is a
significant misclassification of individuals without disease. As the diagnostic threshold is lowered,
an increasing number of patients without disease would be told they have a positive test result,
and by implication, the disease in question. The formula for sensitivity is:
true-positives
× 100
true-positives + false-negatives
The sensitivity of a True-positives and false-negatives are groups with disease; as noted above, sensitivity focuses
laboratory test is its on those with disease.
capacity to identify all
individuals with disease.
Specificity is a statistical The Definition of Specificity of a Laboratory Test
term that indicates the The population of individuals without disease is the focus of specificity. Specificity is a statisti-
effectiveness of a test to cal term that indicates the effectiveness of a test to correctly identify those without disease.
correctly identify those When used to describe a laboratory test, it does not refer to its ability to diagnose a “specific”
without disease. disease among a group of related disorders. One could maximize specificity by raising the
threshold shown in Figure 1–3 to place all those without disease below the line. This would
decrease the number of false-positives because everyone without disease would have a nega-
tive test result. However, there would be a significant misclassification of the individuals with
disease. As the diagnostic threshold is raised, an increasing number of patients with disease
would be told they have a negative test result and, by implication, no disease. The formula for
specificity is:
true-negatives
× 100
true-negatives + false-positives
True-negatives and false-positives are the groups without disease; as noted above, specificity
focuses on those without disease.
The Identification of the Appropriate Value

for the Diagnostic Threshold
For diseases that are serious and treatable, and for which a second confirmatory laboratory test
exists, it is important to maximize sensitivity as in Figure 1–2. For example, for diagnosis of
cannot completely
separate positive and
Test results
negative test results
Line drawn to
maximize specificity––
False-
identifies all those
negatives
without disease correctly
No disease Disease
FIGURE 1–3 A clinical situation in which a diagnostic threshold is selected to maximize specificity.
cannot separate
False- test results
Test results
positives
Line drawn to
minimize false
False- positives and
negatives false negatives to
identify the greatest
number of patients
correctly
No disease Disease
FIGURE 1–4 A clinical situation in which a diagnostic threshold is selected to minimize the
number of false-positives and false-negatives.
AIDS, it is better to have a few false-positives that can be subsequently correctly identified with
a confirmatory test than to fail to identify individuals with HIV infection who might unknow-
ingly infect others. However, for diseases that are serious and not curable, a false-positive result
is catastrophic for the patient. For such diseases, such as pancreatic cancer, it is better to use the
threshold shown in Figure 1–3 for diagnosis because if individuals with disease are missed, it will
have no effect on the treatment or outcome. When there are no compelling reasons to maximize
either sensitivity or specificity, the threshold value should be established to minimize the total
number of false-positives and false-negatives, as shown in Figure 1–4.
The Definition of Predictive Value of a Positive Test

The population of individuals with a positive test result is the focus of positive predictive value.
The positive predictive value for a laboratory test indicates the likelihood that a positive test result
identifies someone with disease. It should be noted that the predictive value of a positive test is
greatly influenced by the prevalence of the disease in the area where testing is performed. As The positive predictive
an example, a screening test for HIV infection is more likely to be confirmed as positive in an value for a laboratory test
area where many individuals are infected with HIV, as opposed to a location where there is only indicates the likelihood
a rare case of HIV infection. In the latter situation, most of the positive HIV tests in the initial that a positive test result
evaluation of a patient are found to be false-positives by confirmatory tests. A high percentage of identifies someone with
false-positives from a low prevalence disease, as shown in the following formula, decreases the disease. The negative
predictive value of the positive test: predictive value for a
laboratory test indicates
true-positives the likelihood that a
× 100 negative test result
true-positives + false-positives identifies someone
True-positives and false-positives are the groups with a positive test result; as noted above, without disease.
positive predictive value focuses on those with a positive test.
The Definition of Predictive Value of a Negative Test

The population of individuals with a negative test result is the focus of the negative predictive
value. The negative predictive value for a laboratory test indicates the likelihood that a negative
test result identifies someone without disease. It is not greatly influenced by the prevalence of
disease because false-positives are not included in the formula for negative predictive value. The
formula for predictive value of a negative test result is:
true-negatives
× 100
true-negatives + false-negatives
True-negatives and false-negatives are the groups with a negative test result; as noted above,
negative predictive value focuses on those with a negative test result.
Excellent precision Excellent precision

Poor accuracy Excellent accuracy
Poor precision Poor precision

Excellent accuracy Poor accuracy
FIGURE 1–5 A series of “bulls-eye” illustrations that display excellent or poor precision
and accuracy.
The Difference Between Prevalence and Incidence

The prevalence of a disease reflects the number of existing cases in a population. It is usually
expressed as a percentage of a certain population. Incidence refers to the number of new cases
occurring within a period of time, usually 1 year. For example, in the United States, sore throat
has a low prevalence because considering the size of the population there is a low percentage of
individuals at a given time afflicted with sore throat. However, it has a high incidence because
many new cases of sore throat appear each year.
Precision refers to the Precision versus Accuracy

ability to test 1 sample Precision refers to the ability to test 1 sample and repeatedly obtain results that are close to each
and repeatedly obtain other. This does not infer that the mean of these very similar numbers is the correct number (see
results that are close to Figure 1–5). Some analyses, which have great precision, are very inaccurate. The accuracy reflects
each other. This does
the relationship between the number obtained and the true result. Thus, a sample could have high
not infer that the mean
accuracy but low precision if it provides the correct answer but has substantial variability as the
of these very similar
sample is repeatedly tested.
numbers is the correct
number.
Analyzing Errors in Laboratory Performance
There are 3 phases of laboratory analysis. The first of these is the preanalytical phase. This time
frame is from patient preparation for the laboratory test, through the time of sample collection,
until the sample arrives in the laboratory. Most of the errors in laboratory test performance occur
in this phase. Examples of preanalytical errors are: inappropriate preparation of the patient, such
as not fasting for a particular test in which fasting is required; ingesting drugs that will interfere
with the laboratory tests; collection of the specimen in the wrong tube; delayed transport of the
specimen to the laboratory; storage of the sample at an incorrect temperature; and collection of
an inadequate amount of blood in vacuum tubes containing a fixed amount of anticoagulants. All
these errors occur before the sample arrives for analysis and make it impossible, no matter how
great the analytical precision within the laboratory, to provide a test result that truly reflects the
patient’s condition. The second phase is the analytical phase, which is the time that the sample
is being analyzed in the laboratory. Errors can occur during this process, but they are much less
common now because of the high level of automation of many laboratory instruments. Examples
of analytical errors are: incorrect use of the instrumentation and the use of expired reagents. The
third phase of laboratory test performance is the postanalytical phase, which begins when the
result is generated and ends when the result is reported to the physician. Example of errors in this
phase, which are more common than analytical errors but less common than preanalytical errors,
are: delay in time to enter a completed result into the laboratory information system and report-
ing results for the wrong patient.
Minimizing Errors in the Selection of Laboratory Tests

As the number of laboratory tests has increased in size, complexity, and cost, health care providers
are highly challenged to select the correct tests, and only the correct tests, in pursuit of a diagnosis.
One approach commonly implemented to assist physicians in correct test selection is the use of a
reflex test algorithm. Tests are ordered by algorithm selection, such as selection of an algorithm to
determine the cause of a prolonged PTT result. Using the algorithm, the clinical laboratory notes
the results of the first test in the algorithm, and that result determines which test is performed
next. For example, if the prolonged PTT is further evaluated with a PTT mixing study, a normal
result would direct testing toward assays for factors VIII, IX, XI, and XII. An elevated result in the
PTT mixing study, on the other hand, would direct testing toward an inhibitor in the PTT reac-
tion, such as a lupus anticoagulant. Testing is continued within the algorithm until a diagnosis,
which explains the prolonged PTT in this case, is identified.
Laboratory test selection is also made more difficult because many laboratory tests have syn-
onyms, and many compounds have related forms. For example, the test most commonly known
as the lupus anticoagulant is also called the lupus inhibitor, and the general term that includes
the lupus anticoagulant and related entities is antiphospholipid antibody. Vitamin D has several
isoforms that include 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Incorrect laboratory
test selection is a major source of medical error.
Minimizing Errors in the Interpretation of Laboratory Test Results

With thousands of tests on the clinical laboratory test menu, it is impossible for a health
care provider to understand the clinical significance of an abnormality for each test. This has
become particularly noteworthy with the introduction of tests for genetic alterations, because
there are so many and the clinical significance of the alterations may not yet be well established.
In some institutions, narrative interpretations of complex clinical laboratory evaluations are
prepared by experts in the field. In most institutions, such narratives require a special request
for completion, but an emerging concept is to provide narrative interpretations for all complex
clinical laboratory evaluations automatically, as they are provided in radiology and in anatomic
pathology. Misinterpretation of laboratory test results has been increasingly noted as a source of
poor patient outcome.
PREANALYTICAL VARIABLES THAT

AFFECT LABORATORY TEST RESULTS
The Effect of Age on Laboratory Tests
There are a number of laboratory tests that have different normal ranges for patients of differ-
ent ages. This is particularly important in pediatrics. Newborns especially have many different
normal ranges than adults or older children for substances in blood and other bodily fluids. For
example, several of the coagulation factors do not reach adult levels for many months after birth.
As a second well-known example, the cholesterol level rises with age.
The Effect of Gender on Laboratory Tests

Gender has a significant bearing on many laboratory tests. Testosterone and estradiol are obvious
examples. In addition, among women there are variations in the serum concentration of various
hormones throughout the menstrual cycle.
The Effect of Body Mass on Laboratory Tests

Muscle mass can affect the level of certain compounds, such as creatine kinase, in the blood. It is
also known that there is an increase in the serum cholesterol level with obesity, because the cho-
lesterol level is related to the amount of body fat.
One of the most Preparation of the Patient for Laboratory Testing

commonly encountered For certain laboratory tests, there are a number of special preparations of the patient that are
patient preparations is necessary to provide the most clinically useful, accurate, and precise result. One of the most com-
fasting, usually for 8 to
monly encountered patient preparations is fasting, usually for 8 to 12 hours, depending on the
12 hours, depending
test. The serum triglyceride level can be significantly affected by eating, and fasting is absolutely
on the test. The serum
required. Another test for which fasting is required is the fasting blood glucose used in the evalu-
triglyceride level can be
significantly affected ation of a patient for diabetes.
by eating, and fasting
is absolutely required.
Another test for which
Patient Posture for Blood Collection
fasting is required is the Patient posture may affect the result for certain tests. There is a lower plasma volume when the
fasting blood glucose. patient is upright because there is pooling of fluid in the dependent parts of the body when stand-
ing. When the patient is supine, there is a movement of fluid back into the circulation from the
tissues. The extra volume in the circulation can dilute certain compounds in the blood. It is best
to monitor the patient in the same postural position if the test result is affected by posture and if
the values need to be compared with one another over time.
Differences in Test Results Between Samples

of Venous, Arterial, and Capillary Blood
Venous blood may have a different concentration of a compound than arterial blood. The best
examples are the blood gases that show marked differences between arterial and venous blood
because of the exchange of gases in the lungs. There may also be a difference between capillary
blood and arterial and venous blood. Blood glucose values may differ significantly in capillary
(finger-stick) samples from venous or arterial blood.
INTERFERENCES IN LABORATORY TESTS

Analytical Interferences in Laboratory Testing
Interferences may result in falsely high or falsely low values, depending on the interfering sub-
stance and the particular test. Although there are many compounds that can interfere with the
accurate and precise quantitation of a compound, there are 3 major interferences that must be
considered when selecting and interpreting results of laboratory tests. These are hemolysis that
makes plasma and serum red; elevated bilirubin that makes plasma and serum shades of orange,
green, or brown; and lipemia that makes plasma and serum milky white. There are many drugs,
There are 3 major particularly those that color the plasma and serum, that can produce significant analytical inter-
interferences that must ference. Many automated laboratory tests are spectrophotometric, and therefore depend on mea-
be considered when surable changes in the color of plasma or serum after a chemical reaction. This is why alterations
selecting and interpreting in the color of the serum or plasma often interfere with laboratory test performance.
results of laboratory
tests. These are hemolysis
that makes plasma and Impact of Drugs on Laboratory Test Results
serum red; elevated
bilirubin that makes Drugs can affect laboratory tests in 2 ways—as an interfering substance in the laboratory test
plasma and serum shades only and by producing an effect in the body that alters a laboratory test result. For example,
of orange, green, or there are many drugs that will increase the PT in patients receiving warfarin (coumadin) by an
brown; and lipemia that in vivo potentiation or diminution of warfarin-induced anticoagulation. There are a number of
makes plasma and serum drug effects, however, that alter the result of a particular test strictly in vitro, and do not change
milky white. anything in vivo.
TEST SELECTION GUIDELINES

The Use of Screening Tests Before Esoteric Tests
Screening laboratory tests are typically inexpensive, easy-to-perform assays that indicate whether
additional tests need to be performed to reach a diagnosis. Whenever possible, if a screening
test is available, it should be used before the more expensive or time-consuming tests are per-
formed. An example of the use of a screening test is the partial thromboplastin time (result within
minutes/hours and at low cost) to assess a major portion of the coagulation cascade. Only if
this value is elevated should tests be performed for PTT-related coagulation factor deficiencies
(results within several hours and at high cost).
The Danger of Ordering Too Many Laboratory Tests

As noted in the discussion of the normal range, 5% of individuals who have no disease can fall
outside of the reference range established by the central 95% of healthy individuals. Thus, if an
individual without disease has 20 different tests, it is likely on a statistical basis that he/she will
have 1 abnormal value (5% = 1 of 20). In medical practice, the abnormal test result for the normal
patient often leads to further evaluation and raises suspicion for a disease that does not exist.
Thus, by limiting the number of tests ordered for a patient to those relevant to the clinical presen-
tation of the patient, one is less likely to encounter false-positive or false-negative results.
SPECIMEN PROCESSING AND HANDLING

The Importance of Turnaround Time
An accurate and precise laboratory test result provided after a decision has been made regarding
patient management is of no value. Since results for all laboratory tests cannot be provided imme-
diately, the physicians and laboratory personnel must decide on clinically relevant turnaround
times for each laboratory test. In addition, if a patient is not discharged from the hospital because
of a delay in laboratory testing, this may have a significant financial impact from unnecessary
length of stay. All steps related to turnaround time, from ordering of the test to the reporting of
the result, must be carefully analyzed and shortened as much as possible.
Tubes for Blood Collection

There are a number of different tubes into which blood may be collected. The tubes used for the
vast majority of collections contain a vacuum to help draw the blood into the tube. The tops of
the tubes have a different color depending on the contents of the tube prior to blood collection
(Table 1–1). Several of the tubes contain anticoagulants to prevent the clotting of the blood in the
tube. Clotted blood that is centrifuged to remove the clot and any cells is known as serum. Blood Clotted blood that is
that has not been clotted and is then centrifuged to remove any cells is known as plasma. For centrifuged to remove
many laboratory tests, the same result is obtained in an assay if serum or plasma is used. However, the clot and any cells is
this is often not the case. The clotting of the blood, for example, makes blood cell counts and coag- known as serum. Blood
ulation tests impossible because the clotting factors are consumed in the clot and the blood cells that has not been clotted
become trapped in it. If the clotting of the blood to form serum is not absolutely necessary, tests and is then centrifuged to
can be performed with a shorter turnaround time using plasma because there is no requisite time remove any cells is known
to wait for the blood clot to form. The amount of anticoagulant in the light blue-top tube must as plasma.
be in a specific proportion to the blood volume in the tube, usually 9 parts blood to 1 part citrate
solution. When an inadequate amount of blood is collected into a blue-top tube, the ratio of blood
to anticoagulant is less than 9:1. This can result in spuriously high values for the PT and PTT tests.
Thus, light blue-top tubes must be filled appropriately to obtain accurate results for clotting tests.
Timing of Blood Collection

Patients may have a need to present for phlebotomy at a certain time of the day if the parameter
being measured has a diurnal variation in its concentration.
TABLE 1–1 Cap Color and Contents of Tubes for Blood Collection
Cap Color Contents
Red Nothing—the sample clots and the product is serum
Light blue Citrate anticoagulant
Purple (lavender) EDTA anticoagulant
Green Heparin anticoagulant

Red/green with gel No anticoagulant, but a gel is present that separates the serum
or plasma and the cells after centrifugation
Gray Fluoride (glycolysis inhibitor for optimum glucose measurements)

with oxalate anticoagulant
Yellow Acid-citrate-dextrose solution (ACD) that anticoagulates the blood

and helps preserve the blood cells during processing
Dark blue Nothing—but the tube is specially treated to permit accurate measurement
of trace heavy materials
Dynamic tests involve the measurement of a patient response to a treatment or stimulus,

and timing of collection is important in these studies. The oral glucose tolerance test, in which
plasma glucose levels are measured after the oral ingestion of a glucose solution, is an example
of such a test.
A third situation in which timing of sample collection is important is in therapeutic drug
monitoring. The serum level of certain drugs is measured to determine if the concentration is
within the therapeutic window. The serum level of a drug varies greatly as the drug is absorbed,
distributed, and metabolized, so the timing of collection must be consistent. For the monitoring
of many drugs, a “trough” level is obtained just before the next dose of the drug is administered.
EFFECTS OF CELL INJURY AND INFLAMMATION

ON SELECTED LABORATORY TESTS
The Release of Plasma Markers of Organ
Damage from Injured Cells
When cells are injured, components of the cells can leak out of the damaged or dead cells and
make their way into the systemic circulation. This permits the measurement of these “marker”
compounds in the serum or plasma as a test for injury to the organ. The most important features
of plasma markers of cell injury are that: 1) they are not rapidly removed from the circulation; 2)
they are relatively organ specific so that the damaged organ is identified; and 3) the compound is
precisely and accurately measured in the clinical laboratory. A well-known example includes the
release of the creatine kinase-MB fraction and troponin from myocardial cells injured by isch-
emia in myocardial infarction.
Markers of Inflammation and the Acute-phase Response

When cells are injured,
The concentration of many plasma proteins changes significantly in patients with inflamma-
components of the
cells can leak out of
tion. Infections (even minor viral illnesses), autoimmune disorders, and many other conditions
the damaged or dead result in an increased concentration of proteins known as acute-phase reactants. Commonly
cells and make their used tests to assess the severity of inflammation, from whatever cause, are the erythrocyte sedi-
way into the systemic mentation rate (ESR) and C-reactive protein (CRP). Examples of acute-phase reactant proteins
circulation. This permits include fibrinogen, which can rise as much as 10-fold over baseline, and von Willebrand factor,
the measurement of these which can rise 2- to 3-fold over baseline. The rise in von Willebrand factor with inflammation
“marker” compounds in can mask a deficiency of von Willebrand factor in patients with von Willebrand disease, and this
the serum or plasma as a highlights the need to obtain baseline values after an acute-phase response subsides for accurate
test for injury to the organ. diagnosis.
The Serologic Diagnosis of Infectious Disease

It is not uncommon to suffer an infection with an organism that is not identifiable by Gram stain-
ing or other microscopic analysis and is not readily cultured. For these infections, the diagnosis
is often made by identifying and measuring the amount of antibody produced in response to an
antigen derived from the infectious agent. The antibody response typically takes several days to
a week or 2 (dependent on past exposure) to emerge, and the appearance of IgM antibody before
IgG occurs in most infections. This is why the presence of IgM antibody in a serologic test is likely
to reflect an acute infection rather than past exposure. Serologic tests may also be designed to
detect and measure an antigen associated with the infectious agent. This obviates the inherent
delay in diagnosis of the infection of up to approximately 2 weeks while waiting for the antibody
response to occur.
REFERENCES
Gornall AG. Basic concepts in laboratory investigation. In: Gornall AG, ed. Applied Biochemistry of Clinical
Disorders. Philadelphia, PA: Harper and Row ; 1980 [chapter 1].
Laposata M, Dighe AS. “Pre-pre” and “post-post” analytical error: high incidence patient safety hazards
involving the clinical laboratory. Clin Chem Lab Med. 2007;45:712–719.
Laposata ME, Laposata M, Van Cott EM, Buchner DS, Kashalo MS, Dighe AS. Physician survey of
a laboratory medicine interpretative service and evaluation of the influence of interpretations on
laboratory test ordering. Arch Pathol Lab Med. 2004;128:1424–1427.
McPherson RA. Laboratory statistics. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and
Management by Laboratory Methods. 21st ed. Philadelphia, PA: WB Saunders; 2006 [chapter 9].
Passiment E, Meisel JL, Fontanesi J, Fritsma G, Aleryani S, Marques M. Decoding laboratory test names:
a major challenge to appropriate patient care. J Gen Intern Med. 2013;28:453–458.
C H A P T E R
Methods
Michael Laposata, James H. Nichols,
Paul Steele, and Thomas P. Stricker
CHAPTER OUTLINE
2
Methods in Clinical Immunology Apheresis 41
Antinuclear antibody (ANA) testing 15 Western blot 42
Protein electrophoresis (PEP) 16 Methods in Clinical Chemistry
Immunofixation to identify monoclonal and General Methods
immunoglobulins 17 Electrolyte measurements: sodium—Na,
Flow cytometry for identification potassium—K, chloride—Cl 43
of cell type and assessment for cell Assays measuring concentration by
surface markers 18 spectrophotometry 44
Nephelometry for quantitation of selected Blood gas measurements 45
proteins and other compounds 19 Urinalysis 46
Cryoglobulin analysis 20 Enzyme-linked immunosorbent assay
Methods in Microbiology (ELISA) 47
Gram stain 21 Fully automated immunoassay with all
Microbiologic culture and organism reagents in solution 48
identification 22 Latex agglutination 49
Blood cultures 23 Chromatography for separation,
Antimicrobial sensitivity tests 24 identification, and quantitation of
Direct and indirect immunofluorescence substances in biologic fluids 50
for antigen detection 25 Mass spectrometry for molecular
Methods in Hematology identification 51
Counting of blood cells with automated Newborn screening by liquid chromatography/
white blood cell differential count 26 mass spectrometry (LC-MS) 52
Peripheral blood smear analysis 27 Point-of-care Testing Methods
Sickle cell screening assay 28 Point-of-care glucose testing 53
Hemoglobin electrophoresis 29 Point-of-care immunoassay
Erythrocyte sedimentation rate 30 on test strip 54
Methods in Coagulation Methods Involving Testing for
The PT and PTT assays 31 Genetic Changes
PT and PTT mixing studies 32 The Karyotype: evaluation of
Coagulation factor assays 33 chromosomes 55
von Willebrand factor assays 34 Polymerase chain reaction with restriction
Platelet aggregation 35 enzyme digestion for detection of
Methods in Transfusion Medicine mutations 56
and Blood Banking Comparative genomic hybridization
ABO/Rh typing 36 (CGH) 57
Blood component preparation 37 Small format genotyping 58
Blood crossmatch 38 Single-nucleotide polymorphism (SNP)
Direct antiglobulin test (DAT) 39 identification by array 59
Indirect antiglobulin test (IAT) 40 Next-generation sequencing 60
N
o textbook in laboratory medicine would be complete without a description of methods
used in the clinical laboratory. The methods described in this chapter are predominantly
the common ones found in clinical laboratories. Each method description provides
an overview of the basic concept of the assay, minimizing the details, while including clinically
important information and a comment on the expense of the test and the complexity of the assay
in the laboratory.
13
14 CHAPTER 2 Methods
Some methods describe specific assays used almost exclusively in the clinical laboratory. For
example, the PT and PTT are tests used for clinical assessment, with the goal to identify factor
deficiencies in the coagulation cascade. Other methods are standard techniques used inside and
outside the clinical laboratory. As an example, flow cytometry is a standard technique used in a
variety of settings, and in this chapter it is shown how it is used in clinical laboratory testing.
There is rapid growth of testing in the clinical laboratory for genetic alterations. These labo-
ratory studies are often highly complex and very expensive, and it is difficult for most clinical
laboratories to perform them for these reasons. In addition, the clinical impact of many genetic
alterations remains unknown. Therefore, it is not uncommon for a genetic test, especially an assay
involving gene sequencing, to produce results that have no known clinical significance. Several
genetic methods are illustrated in this chapter.
Some tests are performed outside the laboratory in the vicinity of the patient. These tests are
called “point-of-care tests,” commonly abbreviated as POCT. This section contains illustrations
for point-of-care testing for glucose and for point-of-care testing for a variety of compounds that
can be detected by immunological methods (immunoassays).
The expense assessment attached to each assay described in this chapter is an approxima-
tion, listed as low, moderate, or high. It should be understood that the charge for the test set by
the institution operating the laboratory is usually proportional to the expense of the reagents,
supplies, and labor required to perform the test. On occasion, however, there is a great disparity
between the actual expense to perform the test and the amount charged by the institution for
the assay. With this in mind, the expense estimation provided for each method in this chapter is
more closely related to the actual cost of reagents, supplies, and labor in the laboratory, with the
understanding that the amount charged for the test should be in the same range of low, moderate,
or high—but it is not always the case.
Each method also has a descriptor to reflect whether it is manual, semiautomated, or highly
automated. A comment has been added if microscopy is involved, as this makes any technique
highly manual. It should be noted that for some methods, there is an option for manual perfor-
mance or for using some level of automation. Manual methods are often less expensive. There is
usually greater automation in the larger clinical laboratories because larger laboratories are more
likely to have the test volume and the financial resources to justify the automated option. The term
semiautomated indicates that there is a manual component associated with the use of an instru-
ment that performs some steps of the analysis.
The turnaround time for an assay is not provided because it is impossible to know all of the
elements associated with the turnaround time for a test within an individual institution. Broadly
speaking, the turnaround time is shorter for assays that are highly automated and less expensive,
and longer for assays that are manual and highly expensive. It is important to understand that
the turnaround time for an assay can be calculated using different starting points. For example,
1 starting point is the time a sample is collected. Another starting point is the time that a sample
Broadly speaking, the enters the laboratory. However, the most relevant starting time clinically, which predates the pre-
turnaround time is vious 2 starting times, is the time at which the physician orders the test. Similarly, there are differ-
shorter for assays that are ent end points in the assessment of turnaround time. Most commonly, the end point is the time
highly automated and at which the result is reported by the laboratory into the laboratory information system. However,
less expensive, and longer it is most important to know when the physician becomes aware of the result. This end point is
for assays that are manual extremely difficult to ascertain, and, therefore, virtually always the end point is considered to be
and highly expensive. the time at which the result is reported by the laboratory.
Finally, it should be noted what methods are not presented in this chapter. There are a number
of methods that have been used progressively less over time, and in many institutions these assays
are no longer performed at all in the clinical laboratory. These are numerous and include the
radioimmunoassay (RIA), immunoelectrophoresis, lipoprotein electrophoresis, and the bleeding
time. Also less frequently performed assays are not described in this chapter. Though this number
of methods may be large, the number of tests performed using these methods account for a small
percentage of the total tests performed in a typical hospital clinical laboratory.
CHAPTER 2 Methods 15
Antinuclear antibody (ANA) testing
Expense: Low Manual with microscopic evaluation
Antinuclear
antibody in
patient serum
Cells on glass slides
incubated with patient
serum—with or without
antinuclear antibodies
If antibodies are present,
they bind to nucleus
Antibody binding is
detected by adding
fluorescent labeled
anti-IgG antibodies
Fluorescent labeled
antibody reveals
patient’s antinuclear
Fluorescent staining antibody
of nucleus can be
homogeneous over
the nucleus, stain the
rim of the nucleus, stain
the nucleoli, or produce
a speckled stain of the
nucleus.
If an antibody is detected, the patient’s serum is progressively diluted until the staining
is no longer detected. The final result includes the highest serum dilution producing a
detectable response and the pattern of nuclear staining.
FIGURE 2–1
Protein electrophoresis (PEP)
Expense: Moderate Semiautomated
Sample can be: – +

Serum for SPEP analysis
Urine for UPEP analysis
Cerebrospinal fluid (CSF)
Urine and CSF are usually

concentrated prior to Sample placed During electrophoresis,
testing to increase the onto agarose gel proteins migrate within
concentration of proteins gel to different locations
in sample
A sample with an additional monoclonal

protein, which can appear in multiple
myeloma, for example, shows a dense Area of gel:
band of protein not present in a sample
from a healthy individual Beta 2 Alpha 2
Beta 1 Alpha 1
Gamma Albumin
Other proteins
Prominent
Broad band of IgG
albumin
immunoglobulins
band
Bands of proteins are generated by
electrophoresis and made visible
by staining the gel
Normal
serum
Serum
from patient
Monoclonal
protein
FIGURE 2–2
Immunofixation to identify
monoclonal immunoglobulins
Multiple aliquots of same sample

onto an agarose gel
Patient specimen is serum

or urine most often, and
occasionally cerebrospinal
fluid (CSF)
Origin
Proteins separated in
gel in individual lanes
Antibody Detects
specificity
IgM
μ kappa or
lambda
IgG Antibodies soaked into strips
ϒ kappa or are overlaid onto each lane
lambda
IgG A
α kappa or Antibody
lambda λ
IgM λ κ
λ IgG λ μ
IgA λ
α
IgM K
κ IgG K ϒ
IgA K This patient has an IgA λ
monoclonal immunoglobulin
FIGURE 2–3
Flow cytometry for identification of cell type and

assessment for cell surface markers
Expense: High Much manual processing with

moderately complex instrumentation
For identification For assessment of cell

of cell type surface markers
F1
Cells flow in
a single
stream
F2
Cell suspension
mixed with
antibodies to
different cell
surface markers —
each of which
has a unique
Laser From amount fluorescent
beam of light label ( F1 is
of light scattered different from F2)
F1
onto cell forward
and to the
side, cell size,
shape, and F2
granularity
determined —
leading to
identification
of cell type
As cells flow in a stream within the instrument and are exposed to laser
light, each fluorescent compound can be identified — fluorescent cells
are positive for the cell surface marker with the specific fluorescent
antibody to that surface marker
FIGURE 2–4
Nephelometry for quantitation of selected

proteins and other compounds
Sample of any body fluid is When the compound is

incubated with an antibody present, antigen–antibody
to the compound being complexes form
measured
Antibody to the
compound is the Antigen is compound
reagent added to being measured
the sample
The amount of scattered Antigen–antibody complexes

light is proportional to the scatter light from a beam
amount of compound being of light shown through the
measured sample
FIGURE 2–5
Cryoglobulin analysis
Expense: Moderate Highly manual method
Cryoglobulins are proteins which

precipitate out of serum at a 37°C
temperature < 37 °C
Therefore, all specimen transport and < 37°C

processing steps must be performed at
37 °C or the cryoglobulin may precipitate out
of serum unintentionally prior to analysis Cryoprecipitate
Patient
Sample split into 2 separate tubes
serum
and both placed at 4° for 1–3 days
at 37 °C
Cryoglobulin in this Tube used to

tube processed by measure a packed
electrophoresis “cryocrit” at 72 hours
Monoclonal Mixed
Polyclonal
immunoglobulins monoclonal
immunoglobulins
only and polyclonal
only
immunoglobulins
Cryoglobulinemia Cryoglobulinemia
type I Cryoglobulinemia
type III
type II
FIGURE 2–6
Gram stain
Expense: Low Manual with microscopic

analysis required
Patient specimen is The material on the

any sample that can glass slide from the
be applied to a glass patient is dried and
slide fixed
The slide is ultimately

subjected to 2 stains
Nonbacterial cells,
Gram positive Gram negative such as white blood
organisms organisms cells, show staining
stain purple are red characteristics of
the cell type
The color, shape, and arrangement of bacteria

are described in the microscopic analysis
“Gram positive cocci in clusters” is one possible
observation in review of a gram stain
Purple, circular bacteria

in clusters, not chains,
are shown here
FIGURE 2–7
Microbiologic culture and organism identification
Expense: Moderate to Mostly manual with much

high, depending on visual inspection of colonies
the extent of the evaluation in different culture media
The sample to be processed can be:

Liquid—such as Solid or On a swab
Sample body fluids other semisolid— from an
collection than blood, which such as sputum, infected site—
is processed stool, or tissue such as a
differently wound
The sample can be:

Growth of Plated onto Inoculated Inoculated
organisms— ≥1 agar plate into a broth onto agar
in aerobic or to permit which promotes within a
anaerobic organisms to growth of tube which
environments grow into microorganisms promotes
colonies the growth
of certain
bacteria
Colonies growing on agar surfaces are first

characterized by colony morphology which
Isolation of
provides an early clue to organism
organisms
identification—and then colonies of interest
can be subcultured for species identification
Microorganisms originating from an isolated

Identification colony can be tested in a panel of
of organisms biochemical tests—the results of which identify
the microorganism with a percent likelihood
FIGURE 2–8
Blood cultures
Expense: High In most laboratories it

is now highly automated
Sample The surface of the arm overlying the venipuncture

collection site must be meticulously cleaned with agents that
eliminate skin microorganisms before venipuncture–
if not, non-pathogenic skin bacteria can contaminate
the blood culture
Blood with or without microorganisms is collected

into bottles for growth in aerobic or anaerobic
environments
Growth of Bottles are placed into specially equipped incubator

organisms for detection of carbon dioxide generated within
individual blood culture bottles
CO2 detected
Positive blood culture–

with growth of microorganisms
generating CO2
Sample from positive blood culture

bottle is then processed for organism
isolation, identification, and
antimicrobial sensitivity
FIGURE 2–9
Antimicrobial sensitivity tests
Expense: High Can be highly manual, as in disc

diffusion method, or semiautomated,
as in dilution method
Microorganisms originating from an isolated

colony are placed in a liquid suspension
Dilution method Disc diffusion method
Organisms into multiple tubes Organisms spread to completely cover a large

agar plate which supports organism growth
More antimicrobial agent into

each sequential tube
Discs with different antimicrobial agents

After incubation, concentration of placed onto agar surface and drug slowly
antimicrobial agent that inhibits diffuses from disc
organism growth is determined
After incubation, agents which inhibit

Minimum inhibitory growth of organisms are identified because
drug concentration bacterial growth is far from the disc
Organism-free zone
Drug
Drug B
A
Organisms Organisms not Drug A is a better antimicrobial

growing growing agent than drug B
FIGURE 2–10
Direct and indirect immunofluorescence

for antigen detection
Expense: Moderate Manual with microscopic evaluation
Direct immunofluorescence Indirect immunofluorescence
F F
Antigen Antigen Antigen Antigen
Fluorescent F labeled antibody Antibody which is not

binds to antigen of interest on fluorescent-labeled binds
a glass slide or other surface to antigen of interest on a
glass slide or other surface
F F
Slides read using a
fluorescent microscope
Antigen Antigen
Fluorescent F labeled antibody

to IgG is added and binds to
antibody previously bound to
antigen
FIGURE 2–11
Counting of blood cells with automated white

blood cell differential count
Expense: Low Highly automated
Purple
Whole blood containing RBC, WBC, and

Dry EDTA platelets in purple top tube containing EDTA
crystals
in tube
Cells flow in a column toward tubes

with aperatures through which blood
cells pass and are counted
Cells passing
through smallest
aperature are
platelets RBC not lysed RBC lysed
Hemoglobin
measured
from
lysed RBC
All but a small percentage
of cells passing through Cells passing through
large aperature are RBC, large aperature are
which are also sized as they WBC–with flow
pass through the aperature cytometry in the circuit
to determine mean to identify WBC types
corpuscular volume (MCV) by size and granularity
Hematocrit or packed RBC volume is calculated from number and size of RBC
FIGURE 2–12
Peripheral blood smear analysis
Expense: Low Smear preparation automated or manual,

followed by microscopic examination
Sample collected into a Drop of blood is applied to a glass

purple top tube containing slide and smeared to spread blood
EDTA cells across slide
Smeared
Drop of blood blood
on slide
Microscopic examination is performed to detect

abnormalities in number or in appearance of:
Red blood cells White blood cells Platelets
The peripheral blood smear is commonly used early in the

diagnostic process to assess a patient for an abnormality
involving circulating blood cells
FIGURE 2–13
Sickle cell screening assay
Expense: Moderate Manual assays, and the sickling test

requires microscopic examination
Sample of blood collected into purple top tube containing EDTA—

two available tests to detect sickle hemoglobin illustrated here
Sickling test— Solubility test—

Blood onto glass slide, followed Blood added to a concentrated
by addition of reducing agent over phosphate buffer solution, followed by
blood droplet RBC lytic agent and reducing agent
Hemoglobin S detected by presence Hemoglobin S detected if buffer

of holly leaf or sickle cells upon becomes turbid because hemoglobin S
microscopic exam is not soluble in this buffer
Tests are positive for patients with:

Hemoglobin SS (sickle cell anemia)
Hemoglobin AS (sickle trait)
Hemoglobin S with another abnormal
hemoglobin (example: hemoglobin SC)
RBC with normal RBC with abnormal morphology Cannot see through the
morphology after addition of reducing agent specimen to visualize black
lines on card behind tube if
hemoglobin S is present
FIGURE 2–14
Hemoglobin electrophoresis
Goal of the test is to identify the hemoglobin

types present in a patient’s red blood cells (RBC)
Blood collected
RBC isolated by RBC lysed and
into a purple—top
centrifugation hemoglobin released
tube containing
and washing from cells
EDTA anticoagulant
Migration of hemoglobins
Hemoglobins separated
in patient RBC compared Gel is stained to
by electrophoresis using
to migration of standard reveal bands of
1 or more electrophoretic
hemoglobins (A, F, S, C) hemoglobin
systems
on gel
This patient has If first electrophoresis

hemoglobins S and is inconclusive,
C or hemoglobin SC additional studies to
identify an abnormal
Standard hemoglobin are
hemoglobins performed until
C S F A hemoglobin type is
established
Origin There are many
hemoglobin variants
and some overlap with
the types used as standards
Hemoglobin types can also be separated by isoelectric focusing and by high

performance liquid chromatography (HPLC)
FIGURE 2–15
Erythrocyte sedimentation rate
Expense: Low Manual or semiautomated
Goal of the test is to measure the height of

sedimented RBC after an incubation, often 1 hour
Whole blood placed

RBC allowed to sediment
in a cylindrical vessel
undisturbed within
with markings to
cylindrical vessel
assess column height
Distance
sedimented
Plasma layer in mm/hr is
erythrocyte
sedimentation rate
RBC Layer
FIGURE 2–16
The PT and PTT assays
Blue
Blood collected into tube—

9 parts of blood per 1 part of
9 parts Blood citrate (4.5 ml blood into 0.5
ml citrate usually)
1 part Citrate
Blue top tube with citrate

anticoagulant in tube Tube centrifuged
and plasma removed
for testing
For PTT:
For PT:
Add activator, partial
Add thromboplastin
thromboplastin and
and calcium
calcium
Time to clot
determined
in seconds
FIGURE 2–17
PT and PTT mixing studies
Expense: Low After samples are mixed, the

testing is highly automated
The goal of the test is to determine if a prolonged PT or prolonged PTT is a

result of ≥ 1 factor deficiencies or an inhibitor of the PT or PTT clotting reaction
The PT or PTT, whichever is

Patient plasma mixed with normal
prolonged, is performed with the
plasma pooled (NPP) from multiple
mixed sample immediately after
donors in equal amounts
mixing and up to 1 hr after mixing
Possible results
The prolonged PT or A prolonged PT or A prolonged PTT (not PT) in

PTT in the patient PTT in the patient the patient plasma shortens
plasma becomes plasma remains toward normal immediately after
normal when mixed prolonged when mixed mixing; when the mixed plasma
with NPP—at all time with NPP—at all time is tested 30–60 minutes after
points after mixing points after mixing mixing, the PTT is prolonged
Most likely a lupus anticoagulant

Most likely a for a PTT prolongation, or a rare Most likely an
factor deficiency factor inhibitor other than a inhibitor to factor VIII
factor VIII inhibitor
Mix Mix Mix

Long
PT or PTT
PT or PTT
PTT
Normal
Time after mixing Time after mixing Time after mixing
FIGURE 2–18
Coagulation factor assays
Expense: Moderate, Highly automated after

due to reagents used dilution and mixing steps
Plasma Sample
Patient + deficient
plasma of mixed
only in plasmas
1 factor
Patient plasma mixed All of the factor being Factor I is fibrinogen

with plasma totally measured is derived and is measured in
deficient in the factor from the patient in the a separate assay
being measured mixed plasma involving thrombin
addition to patient
plasma and measuring
time to clot formation
Factor III is tissue factor
Factor level is and is not measured for
determined using clinical assessment
a standard curve that
relates clotting time to Factor XIII which
amount of factor stabilizes a formed
clot can be assessed
by several different
methodologies
Factors VIII, IX, XI, XII

Factors II, V, VII, X
measured in a PTT-based
measured in a PT-based
assay with clotting activator,
assay with thromboplastin
partial thromboplastin
and calcium
and calcium
FIGURE 2–19
von Willebrand factor assays
Expense: High Test for ristocetin cofactor is largely

manual, and tests for von Willebrand
factor antigen can be semiautomated
Test for von Willebrand Test to assess the amount of

factor function: The ristocetin von Willebrand factor protein:
cofactor assay the von Willebrand antigen assay
Enzyme-linked immunoassay
The amount of von Willebrand (ELISA) and other methods
factor activity is proportional involving antibody to von
to the rate at which fixed platelets Willebrand factor can be used
aggregate in response to ristocetin to quantify amount of von
Willebrand factor protein
Shallow slope
aggregation
indicates slow
% Platelet
Steep slope aggregation

indicates rapid
aggregation
Time Time
Addition of Addition of
ristocetin ristocetin
High von Willebrand Low von Willebrand
factor activity factor activity
FIGURE 2–20
Platelet aggregation
Expense: High Manual test requiring

careful performance to
generate accurate result
Goal of the test is to assess the function of circulating platelets
The sample is centrifuged

Sample collected relatively slowly to sediment
in a blue top tube the larger and more dense
containing citrate white blood cells and red
blood cells from the platelets
Platelets remain
in plasma
WBC and RBC
Platelet activators added

to tubes with platelets—
functional platelets will Free floating
clump and fall to bottom platelets
of tube—poorly functioning
platelets do not
Platelet rich plasma (PRP)
is removed to separate tubes
Result expressed as percent of full

aggregation response, as measured
spectrophotometrically in a platelet
aggregometer
Functional Platelets with

platelets impaired function
FIGURE 2–21
ABO / Rh typing
Expense: Low Automated or manual
Forward typing: Reverse typing:

To detect antigens on RBC To detect antibodies in serum
which can bind to RBC antigens
Add antibodies to A, B, and Rh Add patient serum with or without

antigens in 3 separate tubes anti-A and anti-B antibodies to A
(1 for A, 1 for B, 1 for Rh) positive and to B positive RBC
containing patient RBC (A cells in 1 tube and B cells
in another)
Clumping of RBC Failure to clump Clumping of RBC Failure to clump

indicates presence indicates absence indicates presence indicates absence
of antigen on RBC of antigen on RBC of antibody to RBC of antibody to RBC
antigen on cells antigen
used (either A or B)
FIGURE 2–22
Blood component preparation
Expense: Blood products are expensive; The process of component

separation of whole blood into blood preparation is manual
components is moderately expensive
Anticoagulated whole blood is obtained

Whole
blood from a volunteer blood donor
The bag of whole

blood is centrifuged
Packed Packed RBC are obtained Plasma containing

RBC and stored at 1–6 °C platelets is obtained
The bag of platelet rich

plasma is centrifuged
Platelet concentrate
Fresh Plasma is obtained Platelet
is obtained and stored concen-
frozen
and stored at <18 °C
plasma at 20–24 °C trate
If not maintained as fresh frozen plasma,

by various methods plasma can be used
to prepare cryoprecipitate, immunoglobulins,
albumin, or factor concentrates
Coagul-
Cryo- Immu- ation
precip- noglo- Albumin factor
itate bulins concen-
trates
FIGURE 2–23
Blood crossmatch
Expense: Low Process described below is manual
The goal of the test is to determine if anything in

the blood of a patient recipient will hemolyze or
agglutinate the RBC from a potential donor
Patient serum mixed with RBC Sample checked for hemolysis

from a potential donor, followed or agglutination—either of which
by centrifugation, incubation, makes the potential donor blood
and addition of other reagents incompatible for the patient
Positive for hemolysis or Negative for hemolysis or

agglutination—incompatible agglutination—compatible
unit—do not transfuse unit suitable for transfusion
Agglutination
Intact RBC with
no agglutination
Hemolysis
FIGURE 2–24
Direct antiglobulin test (DAT)
Expense: Moderate Largely manual method
Goal of the test is to determine if IgG immunoglobin or C3d complement

is bound to the surface of the patient’s red blood cells
Suspension of patient’s RBC

placed in 3 separate tubes
RBC + anti- RBC + anti- RBC + anti-C3d

IgG and anti- IgG (performed (performed if
C3d (initial if initial test initial test is
test—detects is positive) positive)
IgG and C3d)
If IgG or C3d is present on RBC, antibody binds to RBC,

resulting in RBC agglutination and/or RBC hemolysis
FIGURE 2–25
Indirect antiglobulin test (IAT)
Expense: Moderate Largely manual method
Goal of the test is to detect antibodies not bound to RBC

present in the plasma or serum of a patient or donor that
can become bound to RBC
To each tube of test sample

Plasma or serum is added different RBC, and
sample being tested each RBC sample is positive
for antibodies to RBC is for certain antigens from the
placed in multiple tubes Rh, MNS, P, LEWIS, KELL,
KIDD, and DUFFY systems
Agglutinated
RBC
Tube without RBC Tube with RBC

agglutination or hemolysis agglutination (or
hemolysis)
The sample tested does The sample tested
not contain an antibody to contains an antibody
the RBC and its associated to an RBC antigen
antigens in this tube in this tube
Additional testing ultimately

reveals the exact RBC antigen
to which the antibody in the
test sample binds
FIGURE 2–26
Apheresis
Expense: Very high Moderately invasive

clinical procedure
Intravenous line returns

Intravenous line takes blood non-collected components
out of patient and replacement fluids
and/or cells
Plasma and cells separated

by centrifugation in
apheresis device within a
sterile circuit
Collection could be for:

Plasma (plasmapheresis)
Platelets (plateletpheresis)
WBC (leukapheresis)
RBC (red blood cell exchange)
Goal of the procedure is to selectively remove

from the patient’s circulation either plasma, platelets,
white blood cells or red blood cells—replacement of
plasma or red blood cells can occur depending
upon the clinical indication for the procedure
FIGURE 2–27
Western blot
Expense: High Manual method
Goal is to identify antibodies Example: Identification of antibodies

in patient serum directed at in serum to proteins within the human
specific proteins immunodeficiency virus (HIV)
Proteins bound to solid

phase—but not stained—
no protein bands visible
If antibody is present which binds to this protein, it will bind
Antibodies from
patient serum
Band of
protein
Antibody binding detected by anti-human immunoglobulin

linked to an enzyme E
Uncolored substrate
E E
E E
Colored product
Protein band with bound

antibody becomes visible
FIGURE 2–28
Electrolyte measurements: Sodium–Na,

Potassium–K, Chloride–Cl
Sample can be plasma or serum—

The sample must be carefully
since plasma must be clotted to
collected, handled, and transported
produce serum, and this increases
to avoid hemolysis—which produces
the time to complete the test, plasma
a spuriously high value for potassium
has become the preferred specimen
Plasma Detection of individual ions by ion

Centrifugation selective electrodes
Ionized (free) calcium, among other
Blood cells
ions, can also be measured with ion
selective electrodes
Whole blood in
anticoagulant
heparin in the tube Plasma can Plasma specimen
be removed for analysis
for analysis
Or Or
Be made to clot Serum specimen

Whole blood in red to produce serum removed from clot
top tube without and blood cells
anticoagulant
Incubation to permit Centrifugation Serum

clot formation
Blood cells
with clot
FIGURE 2–29
Assays measuring concentration by spectrophotometry
Expense: Low Highly automated assays for most

substances or activities measured
Sample is usually patient plasma or serum—for many assays, either can

be used and for others, one or the other is specifically required
Common scenarios for testing and detection:
Patient sample Compound + Reagent A Enzyme 1 Reaction product

containing compound of interest results in a measurable
of interest, which is Or change in light
reacted with added Compound + Reagent A absorbance at a
reagents and of interest certain wave length
enzymes to generate
a product detected Enzyme 1
spectrophotometrically Product X
Product X + Reagent B
Enzyme 2
Product Y
Patient sample Reagent A + Reagent B Enzyme activity is

containing enzyme proportional to the
activity of interest, Enzyme activity amount of product X or
which is detected upon being measured product Y, depending
addition of substrate Product X upon the design of the
for the enzyme assay—and the final
leading to the Or product is measured
generation of a Product X + Reagent C spectrophotometrically
product detected
spectrophotometrically Enzyme 1
Product Y
Reagents A, B and C, and enzymes 1 and 2 are all added to lead to the generation
of a product that is proportional to the compound of interest or reflects the enzyme
activity being measured
FIGURE 2–30
Blood gas measurements
Expense: Low Requires injection of whole

blood sample into instrument
with no additional manipulation
The sample for testing is arterial blood transported on ice
Sample is injected into blood gas analyzer
pH electrode measures pCO2 electrode—a pH pO2 electrode—

blood pH electrode in a sleeve detecting an electric
with bicarbonate— current proportional
measures blood pCO2 to the amount of
oxygen in the sample—
measures blood pO2
FIGURE 2–31
Urinalysis
Expense: Low Can be completely manual or highly

automated—sediment is examined
microscopically which can be aided
by automation in some instruments
There are two parts to a complete urinalysis
Chemical tests Sediment analysis
Reagent pads on a dipstick Urine is centrifuged and the concentrate

change color when the compound is examined microscopically—notable
of interest is present—after a brief findings include:
dip of the stick into the urine RBC
WBC
Reagent pads constructed to Collections of RBC or WBC in casts
measure semi-quantitatively some A variety of cells from the urogenital tract
or all of the following in the urine: A variety of different crystals
identifiable by size and shape
Specific gravity
pH
White blood cells
Nitrite
Protein
Glucose
Ketones RBC RBC cast Epithelial cell
Urobilinogen
Bilirubin
Blood
WBC WBC cast Crystals
FIGURE 2–32
Enzyme-linked immunosorbent assay (ELISA)
Expense: Moderate Semiautomated to

almost fully automated
To detect antibodies in the To detect an antigen in the

patient’s serum patient’s serum
Antibodies are detected by binding to a Antigens are detected by binding to

corresponding antigen fixed to a surface corresponding antibodies fixed to a surface
Antibody in Antigen in
patient serum patient serum
Antigen fixed Antibody fixed

to surface to surface
Detection with antibody to

Detection with anti-human antigen that has an enzyme
antibody linked to an enzyme E E linked to the antibody
E E E E E E E E
Add uncolored substrate for enzyme and enzyme converts it to a colored

product—the darker the color, the more antibody or antigen in the patient serum
Uncolored Colored
substrate product
E
FIGURE 2–33
Fully automated immunoassay

with all reagents in solution
Expense: Low Automated

No antigen in sample—negative test
Example Ag-Enz
as antigen: Ag-Enz
HCG Ag-Enz
Antibody Ag is HCG No HCG

to HCG in specimen
Ag-Enz Ag-Enz
Ag-Enz
Ag-Enz
Binding of Ag-Enz inactivates

enzyme activity
No signal
Antigen in sample—positive test
Free Ag
Ag-Enz Ag-Enz
Ag-HCG
in this Ag Ag-Enz Ag
example Ag
HCG present
in specimen
Ag
Ag Ag-Enz
Ag Ag
Uncolored Colored
substrate product
Free Ag-Enz allows enzyme to be active
Enzyme Enzyme conjugate activity

activity is linearly proportional to
antigen concentration in
sample
Antigen concentration
Enz: Enzyme Ag: Antigen HCG: Human chorionic gonadotropin
FIGURE 2–34
Latex agglutination
Expense: Low Automated or manual
Goal of the test is to detect the presence of a compound

in a patient specimen that visibly clumps latex particles
Patient specimen which contains

molecule of interest
Mix with latex beads
Antibody on beads
binds the molecules
of interest
Latex beads clump Latex beads fail to

when molecule is clump when molecule
present is absent
FIGURE 2–35
Chromatography for separation, identification, and

quantitation of substances in biologic fluids
Expense: High Test requires manual

processing and data analysis
Sample is extracted
to remove extraneous
molecules
Low-molecular-weight Large molecules such as

compounds proteins and nucleic acids
Analysis by
Analysis by high-performance
gas chromatography (GC) liquid chromatography
(HPLC)
Molecules in sample
may require chemical
derivatization to increase
volatility in gas phase
Injection of samples into instrument with column separation
Column temperature Mobile solvents and/or

increased to separate solvent pH altered
compounds to separate compounds
Compounds identified and quantitated

Signal
using flame ionization (GC),

spectroscopy (HPLC), mass spectrometry
(GC and HPLC), or other detection method.
Time
FIGURE 2–36
Mass spectrometry for molecular identification
Expense: High Semiautomated with high complexity

of laboratory instrumentation
Molecular compounds of interest

A patient sample is
are isolated from other molecules
processed to render
in sample by liquid chromatography
it suitable for analysis
or gas chromatography
In mass spectrometer, molecule

is broken into different size mass
fragments creating a “fingerprint”
for the molecule
The fingerprint of the molecule in

the patient specimen is compared
to a large library of molecular
fingerprints
Abundance
Abundance
Mass/Charge Mass/Charge
Fingerprint of a Fingerprint of compound
known molecule in patient specimen
Molecule is identified
because fingerprints
match with very high
percentage of agreement
FIGURE 2–37
Newborn screening by liquid chromatography/mass

spectrometry (LC-MS)
Expense: High for instrumentation; Semiautomated with

moderate for test performance high-complexity instrumentation
After 24 h of life, heel of neonate

is punctured to obtain whole blood—
which is spotted onto a special card
and sent to a central laboratory Blood spots
on card
Molecular compounds of interest LC-MS

are extracted from a portion of the analysis
blood spot and separated by liquid Portion of
chromatography 1 blood spot
A tandem mass spectrometer

identifies the compounds +
of interest and quantifies the Screening
amount of each—and test results
those compounds in higher − −
concentrations than the
reference range are “positive” in the
newborn screening test Compound A B C
Confirmation of positive test True-positive

Additional
for compound C is testing for vs
performed to determine if result compound C
is true-positive False-positive
FIGURE 2–38
Point-of-care glucose testing

Expense: Low After sample is added to test strip,
testing is automated
Result display
A blood sample, which is

commonly from a finger stick,
is applied to a test strip
Test strip Glucometer
with sample
Top-down view of strip

Sample application occurs
onto an absorbent pad
that separates blood cells
Sample Sensors from plasma
application area
is an absorbent pad
Glucose within plasma mixes

Side view of strip with reagents embedded on
RBC, test strip as plasma moves
WBC, toward sensors
and Plasma
platelets passes through Enzyme reactions with glucose
stay on top of absorbent pad as substrate produce electrons
absorbent pad detected by sensors at the
end of the test strip
inserted into glucometer
Top-down view of strip

below absorbent pad
Sensor signal is proportional

to concentration of glucose
Reagents that react with in sample, which is shown
glucose embedded here as in the result display window
plasma moves toward sensors of the glucometer
FIGURE 2–39
Point-of-care immunoassay on test strip
Cost: Moderate Manual test

requiring interpretation
Negative test
Top-down view of strip Side view of test strip: Before application
of sample
Sample Band in
application control Sample Anti-antigen Goat
spot region application— antibodies anti-
blood or fixed to strip mouse
Positive test urine antibodies
Area has
dried mouse anti- fixed to strip
Band that antigen antibodies
shows test as positive with colored conjugate
Negative test
Side view of test strip after sample application—Antigen (Ag)
Fluid migration is absent
with wicking
action out of
sample
application spot Sample Solubilizes No Goat anti-
added mouse antigen mouse antibodies
to well anti-antigen in sample bind mouse anti-
antibodies so– antigen antibodies
with colored to make 1 band in
conjugate control region
Positive test
Side view of test strip—Antigen (Ag) is present
Example antigen Ag
is HCG—in a Ag AgAg
pregnancy test Ag
Mouse anti-antigen Goat anti-mouse

antibodies fixed to strip antibodies fixed to
bind Ag—and the solubilized strip bind excess
mouse anti-antigen solubilized mouse
antibodies with the colored anti-antigen antibodies
conjugate also bind Ag to create a second
to form a colored band colored band—bands
where fixed antibodies shown within dashed lines
Ag: Antigen are imbedded onto strip
FIGURE 2–40
The Karyotype: evaluation of chromosomes
Cost: Expensive Manual test with

detailed interpretation
Human somatic cells have 46 chromosomes with

22 homologous autosomal pairs and 2 sex
chromosomes—XX in females and XY in males
Portion of sample After 16–72 h of

Blood sample placed into tissue incubation time, colcemid
collected in culture medium with added to arrest cell
heparin-containing mitogens to promote division and ethidium
vacuum tube lymphocyte proliferation bromide added to
or without mitogens elongate chromosomes
Slides are stained Nuclei are dropped

Cells are ruptured
with Giemsa stain to onto slide to optimize
while nuclei remain
produce characteristic spreading of metaphase
intact
dark and light bands chromosomes
Microscope or imaging Evaluation for numerical abnormalities

software used to capture and structural rearrangements by counting
metaphase cells and chromosomes and assessing the
enable karyotyping banding pattern for each chromosome
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18
19 46, XY 20 21 22 X Y
A normal male karyotype

(Courtesy of Dr Ferrin Wheeler)
Description of karyotype is total number of chromosomes,
the sex chromosomes, description of any abnormality
Example: 47, XY, +21 is a male with trisomy 21.
FIGURE 2–41
Polymerase chain reaction with restriction enzyme

digestion for detection of mutations
Expense: High Largely manual with increasing

semiautomation of methods
Primers for DNA fragment

DNA is isolated
DNA is denatured of interest allowed to
from whole blood
to separate strands anneal with separated
or tissue
DNA strands
Denaturation, annealing, DNA polymerase

DNA fragments of
and elongation steps is added to
interest accumulate
repeated up to promote elongation
exponentially
30–40 times of DNA strand
To detect mutations in patient DNA,

PCR products can be cleaved with a
DNA cleaving restriction enzyme that
reveals genetic difference
Example: Mutation Example: Mutation

detected by DNA detected by DNA
fragments in fragments separated by
polyacrylamide gel capillary electrophoresis
Wild type
Heterozygous
for mutation
Homozygous
for mutation
FIGURE 2–42
Comparative genomic hybridization (CGH)
Expense: High Mostly manual with some

automated steps
DNA is isolated from DNA from tumor is Tumor/normal DNA is

tumor and normal fluorescently labeled red; combined and hybridized
tissue from the DNA from normal is to a microarray, and then
same individual fluorescently labeled green scanned with a laser
CGH can also be used to identify

congenital chromosomal
Microarray has ~1,000,000
abnormalities. In this case, DNA
probes covering the
from the affected individual is
human genome
labeled red and control DNA from
a normal individual is labeled green
If sequence is present If sequence is amplified in If sequence is deleted in

in tumor and normal, tumor, probe is mostly red tumor, probe is mostly green
color is yellow (most of DNA comes (most of DNA comes
(mix of red and green) from tumor) from normal)
FIGURE 2–43
Small format genotyping—detect single-nucleotide

polymorphisms, mutations, and insertion
or deletion of bases (indels)
Expense: High Mostly manual with
some automated steps
DNA is isolated Primers that flank polymorphisms or

from tissue, mutations of interest (dozens to hundreds)
tumor, blood are used to amplify these regions
A 3rd primer is designed so that it anneals

immediately before the polymorphism; the
next base added to the primer will be at
the polymorphic position
Polymerase and ATCG are added—each of

the four bases is fluorescently labeled with a
different color and polymerase adds 1 base
at the polymorphic position
Sample is run on a capillary sequencer and

the base that was added at the polymorphic
position is detected by a laser
If the patient is homozygous, 1 color If the patient is heterozygous, 2 colors

will be present, and the color present will be present, and the colors present
indicates which base was added indicate which bases were added
G G
C C
G A
C T
FIGURE 2–44
Single-nucleotide polymorphism (SNP)

identification by array
Expense: High Mostly manual with

some automated steps
Genomic DNA is DNA is sheared and DNA is hybridized to

isolated from labeled with a oligonucleotide
blood fluorescent label array
For each SNP (single-nucleotide

polymorphism, position in human
genome known to have different
bases in different individuals),
there are 2 probes, which differ in
sequence only at the polymorphic
position
Unbound DNA is washed away,

and the array is scanned with a
laser—if a polymorphism is
present, the fluorescent labeled
DNA remains bound to the array
and the spot glows
If an individual is If an individual is
TAGC homozygous for a position, heterozygous at TAGC
ATCG only 1 of the probes will a position, both ATCG
glow, as nothing hybridizes probes glow AGGC
ACCG ACCG
to the other probe
In this way, over 2.5 million SNPs can be analyzed on a

single chip—data can be analyzed for both genotype and
copy number variation
FIGURE 2–45
Next-generation sequencing
Expense: High Mostly manual with some
automated steps
Library may be sequenced as
Ligation and PCR are used to is (whole genome sequencing)
add universal adaptors to each or pieces of the genome may
DNA is isolated
piece of DNA. This makes the be selected by hybridization,
from a sample
sequence at the ends of every washing away the unbound
and sheared
piece of DNA the same. This is portions, and then eluting and
called a sequencing library sequencing what remains
(targeted sequencing)
Polymerase + ATCG +
sequencing primer
(which anneals to
DNA library is hybridized to a
universal adaptors) are Billions of DNA library
flow cell, which is coated with
added. Each base is molecules are now
oligonucleotides complemen-
fluorescently labeled. attached to the flow cell
tary to the universal adaptors
Each base is termi-
nated, so only 1 base
can be added at a time Polymerase adds the first base to every piece of DNA
on the flow cell, and then the chip is scanned with a
laser. Every spot, each of which is an individual piece
of DNA from the sequencing library, will glow 1 of
4 colors depending on which base was added
The fluorescent marker and Polymerase + ATCG + sequencing primer

termination are removed are added—Polymerase adds the next
from the added base, which base to every piece of DNA on the flow
now allows another base to cell, and then the chip is scanned with a
be added laser. Every spot will glow 1 of 4 colors
Repeat 200
times
Cycle 1 Cycle 2 Cycle 3
A G G A T C
A C G
C G T G T C
T A G
T A C A G C A C T
Each spot = 1 library molecule. Images are recorded in each cycle, and the color for each
cycle is used to determine the base sequence. There are billions of spots on a flow cell.
Thus, sequence for spot in upper left corner is A G T
FIGURE 2–46 (Reproduced, with permission, from Shendure J, Ji H. Next-generation DNA sequencing. Nat Biotechnol.
2008;26(10):1135-1145.)
C H A P T E R
Autoimmune Disorders
Involving the Connective
Tissue and Immunodeficiency
Diseases
3
Mandakolathur R. Murali
LEARNING OBJECTIVES
1. Learn the common autoimmune diseases involving primarily the connective
tissue.
2. Understand the disorders associated with immune deficiencies and their
underlying pathophysiology.
3. Learn the diagnostic tests required to establish a diagnosis for autoimmune
disorders and for immunodeficiency disorders.
CHAPTER OUTLINE
Systemic Autoimmune Diseases Involving Diseases of the Immune System 74
the Connective Tissue 64 X-linked Agammaglobulinemia 74
Systemic Lupus Erythematosus 64 Common Variable Immunodeficiency 75
Sjogren Syndrome 64 Hyper-IgM Syndrome 76
Systemic Sclerosis/Scleroderma 67 Selective IgA Deficiency 76
Inflammatory Muscle Diseases 67 DiGeorge Syndrome 77
Mixed Connective Tissue Disease 69 Severe Combined Immunodeficiency
Rheumatoid Arthritis 70 (SCID) Syndrome 77
Amyloidosis 70 Deficiencies of Complement
Cryoglobulinemia 73 Proteins 77
T
he immune system is a tightly regulated network that incorporates both innate and
adaptive pathways. The genes regulating the innate system are coded in the germ line. The
innate immune system is not antigen specific. The cells and soluble factors of the innate
system have pattern recognition receptors (PRRs, such as toll-like receptors) to common motifs
on pathogens and altered self-motifs. The motifs on pathogens are called pathogen-associated
molecular patterns (PAMPs). Altered self-antigens include danger-associated molecular patterns
(DAMPs) as found in heat shock protein, and apoptosis-associated molecular patterns (AAMPs)
as found in ds DNA, RNP, and histones. This response is rapid and there is no memory of the
encounter.
The receptors on the T and B cells of the adaptive immune system are antigen or epitope spe-
cific and clonally variable, and their diversity is derived from gene recombination. The cells retain
61
62 CHAPTER 3 Autoimmune Disorders Involving the Connective Tissue and Immunodeficiency Diseases
TABLE 3–1 Systemic Autoimmune Diseases: Diseases Associated With Positive

Test Results for Antinuclear Antibodies (ANA)
Disease % ANA Positive Titer Common Patterns
Systemic lupus erythematosus—active 95–98 High H>S>R
Systemic lupus erythematosus—remission 90 Moderate–high H>S
Mixed connective tissue disease 93 High S>N
Scleroderma/CREST 85 High S>C>N
Sjogren syndrome 48 Moderate–high S>H
Polymyositis/dermatomyositis 61 Low–moderate S>N

Rheumatoid arthritis 41 Low–moderate S
Drug-induced lupus 100 Low–moderate S
Pauciarticular juvenile chronic arthritis 71 Low–moderate S

Note: ANA patterns on Hep-2 cells by indirect immunofluorescent technique (IFA). Patterns: H, homogeneous; S, speckled; R, rim;
C, centromere; N, nucleolar. Titers: high = 1:1280 to 1:5120; moderate = 1:160 to 1:640; low = 1:40 to 1:80.
memory of the encounter and on subsequent engagement with that antigen, the cells exhibit more
rapid and robust responses.
The immune network is tightly regulated by cells and cytokines, and a derangement in this
immune homeostasis can result in immune response to self-antigens as in autoimmunity (failure
of self-tolerance), or failure to recognize pathogens and eliminate them as occurs in immunode-
ficiency syndromes (failure of immunity). The following 2 groups of disorders are the focus of
this chapter: the autoimmune diseases involving the connective tissue and the immunodeficiency
diseases.
Diseases in which immune responses to self-antigens occur in the context of a genetic pre-
disposition to disease expression are called autoimmune diseases. Some involve organ-specific
pathologic autoimmunity such as Hashimoto thyroiditis and celiac disease, and these are dis-
cussed in Chapters 22 and 15, respectively. The autoimmune disorders discussed in this chapter
are systemic diseases with predominant involvement of the connective tissue, manifesting clinical
features including inflammation of the joints, skin, muscles, and other soft tissues (see Tables 3–1
and 3–2 and Figure 3–1).
The immunodeficiency diseases are subdivided into the relatively rare primary and the more
common secondary immunodeficiency diseases. Primary immunodeficiency diseases are a direct
consequence of either structural or functional derangement in the immune network. Secondary
immune deficiency is the manifestation of a primary infectious disease, such as HIV infection; a
malignancy as seen in lymphoma and multiple myeloma; or exposure to a therapeutic regimen
such as immunosuppression or radiation.
TABLE 3–2 Specific Organ Autoimmune Diseases: Diseases Associated With

Positive Test Results for Antinuclear Antibodies (ANA)
Disease % ANA Positive Titer Common Patterns
The following 2 groups Graves disease 50 Low–moderate S

of disorders are the Hashimoto thyroiditis 46 Low–moderate S
focus of this chapter:
the autoimmune Autoimmune hepatitis 63–91 Low–moderate S
diseases involving the Primary biliary cirrhosis 10–40 Low–moderate S
connective tissue and
Patterns: S, speckled. Miscellaneous causes: low titer positive ANA patterns (mostly speckled) have been described in chronic
the immunodeficiency infectious diseases such as infectious mononucleosis, hepatitis C infection, HIV, subacute bacterial endocarditis, and certain
diseases. lymphoproliferative diseases.
CHAPTER 3 Autoimmune Disorders Involving the Connective Tissue and Immunodeficiency Diseases 63
The clinical features of the disease, the morphologic pattern of the ANA test, and
the serum titer of the positive ANA test are established.
If the ANA is positive, the pattern of staining suggests the differential diagnosis. The
results of specific antinuclear antibody tests often establish the diagnosis.
A negative ANA test can occur in rheumatoid arthritis, inflammatory muscle diseases,
and when there are connective tissue manifestations in patients with selected
chronic infectious diseases.
The following is an algorithm for the serologic evaluation of autoimmune

connective tissue diseases.
If diagnosis is unknown and the ANA is positive, the following

test panel is useful:
(a) anti-ds DNA
(b) anti-SS-A (Ro)
(c) anti-SS-B (La)
(d) anti-Sm
(e) anti-U1 RNP
(f) anti-Jo-1
(g) anti-Scl-70
(a) If the ANA is negative, test for anti-SS-A (Ro)

(b) If the ANA is positive, tests for anti-ds DNA,
For SLE anti-SS-A (Ro), anti-SS-B (La), anti-Sm and,
anti-U1 RNP are informative. Anti-ds DNA
titers are useful to monitor disease activity.
For Sjogren (a) A positive ANA is supported by positive test

syndrome results for anti-SS-A (Ro) and anti SS-B (La).
For polymyositis
(a) A positive ANA is supported by a positive
and
anti-Jo-1 test result.
dermatomyositis
For mixed
connective (a) A positive ANA is supported by a positive
tissue disease result for anti-U1RNP.
(a) If the ANA pattern is the speckled or

For Scleroderma centromeric, anti-Scl-70 (anti-topoisomerase 1)
provides additional diagnostic confirmation.
FIGURE 3–1 An approach to the diagnosis of autoimmune disorders involving connective tissue.
SYSTEMIC AUTOIMMUNE DISEASES INVOLVING

THE CONNECTIVE TISSUE
Systemic Lupus Erythematosus
Description
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease, associated with the
production of antibodies to a variety of nuclear and cytoplasmic antigens. The hallmark charac-
teristic is the generation of antibodies to ds DNA. These antibodies complex to these self-antigens,
and the ensuing immune complexes contribute to the inflammation in many organs, particularly
the skin, joints, kidney, and, to a lesser extent, the cardiovascular and nervous systems, lung, and
hemopoietic cells.
The disease is more common in women than in men and usually appears in early adulthood,
although it is seen in children as well. It not only is more common in African Americans than in
Caucasians but also has a more severe clinical phenotype with renal and vasculitic manifestations
in African Americans.
The candidate genes associated with SLE include those coding for complement components
C1q, C4A, C2, activating and inhibitory FcγR, interferon regulatory factor 5 (IRF5), TNF, MHC
class II (DR2 and DR3), and programmed cell death PDCD1, among others.
Table 3–3 summarizes the laboratory evaluation of SLE and Table 3–4 lists the autoanti-
bodies associated with SLE.
Systemic lupus Diagnosis

erythematosus (SLE) is a According to the American Rheumatologic Association criteria for diagnosis of SLE, the diag-
multisystem autoimmune nosis of SLE is made if 4 or more of the following 11 criteria are present at any time during the
disease, associated course of the disease:
with the production of
antibodies to a variety of
nuclear and cytoplasmic Malar rash Flat or raised fixed erythema over the malar eminences and sparing the nasolabial folds
antigens. The hallmark
characteristic is the Discoid rash Erythematous raised patches with adherent keratotic scaling and follicular plugging;
scarring may occur in older lesions
generation of antibodies
to ds DNA. Photosensitivity Skin rash resulting from reaction to light
Arthritis Nonerosive arthritis involving 2 or more peripheral joints that are swollen or tender and
evidence of effusion
Oral ulcers Mostly painless ulcers in the oral cavity and pharynx
Serositis Pleuritis with pleural rub or effusion; pericarditis documented by rub, EKG change, or
pericardial effusion
Renal diseases Persistent proteinuria greater than 0.5 g/day or 3+ on dipstick or presence on RBC,
granular, tubular, or mixed cellular casts
Neurologic Seizures or psychosis in the absence of metabolic or drug-induced causes
Hematologic Any immune cytopenia—RBC, WBC, or platelets
Immunologic Positive anti-ds DNA antibody, positive antiphospholipid antibody, positive anti-Sm
antibody, and false-positive serologic test for syphilis
Antinuclear An abnormal ANA titer by immunofluorescence or an equivalent assay in the absence

antibody of drugs known to be associated with “drug-induced lupus”
Tests utilized in the initial evaluation and subsequent monitoring of patients with SLE are
shown in Tables 3–3 and 3–4 and Figure 3–1.
Sjogren Syndrome
Description
Sjogren syndrome (SS) is a systemic connective tissue disease, more common in women than in
men. Pathologically, it is an autoimmune exocrinopathy involving the lacrimal glands, salivary
glands, and less often the pancreas. The immune inflammation of these glands contributes to the
TABLE 3–3 Laboratory Evaluation of Systemic Lupus Erythematosus (SLE):

General Laboratory Tests
Laboratory Tests Results/Significance
Complete blood Decrease in RBC, WBC, and platelets either singly or in combination suggests the
count and erythrocyte presence of autoimmune cytopenias; serial CBC is useful to monitor bone marrow
sedimentation rate response to immunosuppressive therapy; ESR if elevated is a useful parameter to
(ESR) follow with therapy
Urinalysis and BUN/ Urinalysis is useful to evaluate proteinuria and any cellular sediments and casts;
creatinine 24-hour protein excretion and BUN/creatinine are useful to monitor renal function
Liver function tests For evaluation of possible autoimmune hepatitis; alterations in plasma lipids either
and lipid profile due to disease or as a sequelae of therapy are to be appropriately managed to
prevent cardiovascular complications
VDRL/RPR test for False-positive VDRL test is noted in SLE; a positive VDRL in the absence of syphilis
syphilis (negative RPR) is a diagnostic criterion for SLE
Antinuclear antibody 95%-98% of patients with active SLE have a positive ANA
Complement assay C3, C4, and factor B are useful to evaluate complement activation; CH50 to detect
congenital complement deficiency especially in familial SLE; low complement values
may reflect disease activity
sicca syndrome, with dry eyes (keratoconjunctivitis) and dry mouth (xerostomia) as characteris-
tic clinical features. The disease can be primary or secondary. The primary syndrome is character-
ized by dry eyes, dry mouth, decreased production of tears as tested by the Schirmer test, and a lip
biopsy that demonstrates inflammation of the minor salivary glands. Serologically, patients with
primary Sjogren show a positive ANA, positive SS-A (Ro), positive SS-B (La), and positive rheu-
matoid factor (RF) in the absence of another connective tissue disease. A prospective study of 80
patients with primary SS followed for a median of 7.5 years reported the following frequencies of
clinical manifestations: a) keratoconjunctivitis sicca and/or xerostomia occurred in all patients
and were the only disease manifestations in 31%; b) extraglandular involvement occurred in 25%;
and c) non-Hodgkin lymphoma developed in 2.5%. Secondary SS is clinically similar to the pri-
mary disorder, but it is additionally associated with clinical and serologic features of another con-
nective tissue disease, such as rheumatoid arthritis (RA) or scleroderma.
TABLE 3–4 Autoantibodies and Clinical Associations in Systemic Lupus

Erythematosus (SLE)
Prevalence Pattern on
Antigen Specificity (%) Hep-2 Cells Clinical Associations
ds DNA 40–60 Homogeneous Marker of active disease; titers fluctuate with

disease activity; correlates best with renal disease
SS-A/Ro 40 Speckled, fine Subacute cutaneous lupus (75%), neonatal lupus

with heart block, complements deficiencies and
photosensitivity
SS-B/La 10–15 Speckled, fine Neonatal lupus
Sm 20–30 Speckled, Specific marker for SLE; may be associated with CNS
coarse disease; not useful in monitoring disease activity
RNP (U1 RNP) 30–40 Speckled, Generally coexists with Sm; RNP is a marker
coarse for MCTD
Histones 50–95 Homogeneous 50%-70% in SLE and >95% in drug-induced SLE
Phospholipids 30 None specific Associated with thrombocytopenia, later trimester

(beta-2 glycoprotein abortions, and hypercoagulable states
I antibodies)
Proliferating cell 3 Finely granular Not sensitive but specific (>95%); not seen in RA,
nuclear antigen nuclear staining other connective tissue disease; antibody rapidly
(PCNA) in rapidly diminished by steroids and immunosuppressive
dividing cells drugs; correlates with arthritis
Systemic sclerosis Diagnosis

is characterized by The diagnostic features are revealed by tests that document the sicca features. The dry eyes are
excessive and often evaluated by the Schirmer test. This test is a measurement of tear flow over a 5-minute period.
widespread deposition
Filter paper is allowed to hang from the lateral inferior eyelid and the length of the paper that
of collagen in many
becomes wet is measured. This test is not reliable, as early in the disease there is excessive lacri-
organ systems of the
mation giving a false-negative test. Demonstration of devitalized corneal epithelium due to kera-
body. Pathologically, the
hallmark is the deposition toconjunctivitis is evaluated by rose Bengal or fluorescein stain. The most accurate test is the slit
of altered collagen in the lamp examination of the cornea and conjunctiva. Tests for quantitating salivary secretion are not
extracellular matrix and a standardized and also are not specific to SS. Biopsy of the minor salivary gland in the lower lip
proliferative and occlusive demonstrating focal lymphocytic infiltration is a useful confirmatory test.
small vessel vasculopathy. Table 3–5 summarizes the laboratory tests useful in diagnosis of both primary and
secondary SS.
TABLE 3–5 Laboratory Evaluation for Sjogren Syndrome

Findings in Sjogren Syndrome
Diagnostic tests for dry eyes
Schirmer test <5 mm wet zone on filter paper in 5 minutes.
Rose Bengal dye test Visualization of devitalized areas in cornea
Tear breakup time Measuring breakup time and tear osmolality after installation of fluorescein;
identifies those who respond to anti-inflammatory therapy
Diagnostic tests for dry mouth
Salivary gland scintigraphy Low uptake of radionuclide is specific for SS, but 33% of patients have a
positive test; not a sensitive test
Lower lip biopsy Presence of lymphoid infiltrate around salivary glands is consistent with
disease
Magnetic resonance imaging MRI is superior to ultrasonography and CT studies and is equivalent to
(MRI) sialography; correlates well with salivary gland biopsy
General laboratory tests
Complete blood count Anemia, neutropenia, lymphopenia, and thrombocytopenia may

including differential count suggest immune destruction. Marked lymphocytosis may suggest clonal
proliferation
Serum electrolytes and liver Hypokalemia if associated with renal tubular acidosis, elevated alkaline
Sjogren syndrome elevated function tests phosphatase suggests primary biliary cirrhosis
is characterized by Erythrocyte sedimentation rate Markers for chronic and acute inflammation
immune-mediated and C-reactive protein
destruction of exocrine
Urinalysis Proteinuria, casts imply renal involvement
glands, particularly the
salivary and lacrimal Quantitative immunoglobulins Polyclonal increase is often noted
glands, with secondary (IgG, IgM, IgA)
development of Serum and urine protein Transformation from polyclonal to monoclonal gammopathy implies
keratoconjunctivitis electrophoresis (SPEP and UPEP) evolution to a B-cell lymphoma. Presence of urine Bence-Jones protein
and xerostomia. A and serum free light chains with confirms monoclonal transformation
positive ANA along altered kappa/lambda ratio
with antibodies to SS-A Laboratory tests for autoimmunity
(Ro) and/or SS-B (La)
Antinuclear antibody (ANA) titer Commonest pattern is speckled; titer greater than 1:160; in 75% of patients
is a serologic feature.
Transition from a Antibodies to SS-A (Ro) With ELISA >90% have a positive test
polyclonal rheumatoid Antibodies to SS-B (La) With ELISA >90% have a positive test
factor (RF) positive to a
RF-negative oligoclonal Rheumatoid factor 70% have positive RF
or monoclonal process Cryoglobulins, C3, and C4 Presence of cryoglobulins and low C3 and C4 are found with multisystem
suggests a malignant disease
lymphomatous Anti-ds DNA antibody In 25%-30% of patients with primary SS
transformation.
Systemic Sclerosis/Scleroderma
Description
Systemic sclerosis is characterized by excessive and widespread deposition of collagen in many
organ systems of the body. The hallmark of this pathologic process is the deposition of altered
collagen in the extracellular matrix. The disorder is characterized pathologically by 3 features: 1)
tissue fibrosis; 2) a proliferative and occlusive vasculopathy of the small blood vessels; and 3) a
specific autoimmune response associated with distinctive autoantibody profile.
The immunologic basis is not well understood, but an aberration in TGF-beta-mediated depo-
sition of collagen has been observed. Antibodies to platelet-derived growth factor receptors have
been incriminated in the development of fibrosis. Both the triggering event and genetic predispo-
sition are not well defined. Although the common organ involved is the skin, the gastrointestinal
tract, kidney, lung, and muscles are also affected as the disease progresses. Renal ischemia leading
to hypertension escalates the complications of this disease. Preponderance in females is common.
Clinically there are 4 major subtypes described:
1. Diffuse cutaneous scleroderma with widespread involvement of skin and visceral organs.
2. Limited cutaneous scleroderma, in which the disease is limited to the digital extremities
and face. CREST syndrome is a variant of this entity. The name is derived from its
features—Calcinosis, Raynaud syndrome, Esophageal dysmotility, Sclerodactyly, and
Telangiectasia.
3. Localized scleroderma that affects primarily the skin of the forearms, the fingers, and later
the systemic organs.
4. Overlap syndromes with features of RA or muscle involvement.
Diagnosis
Ninety percent to 95% of all patients with scleroderma have a positive ANA test. The most com-
mon pattern is finely speckled, followed by centromeric and nucleolar patterns. The ANA activity
is directed against DNA topoisomerase (also known as Scl-70). A definitive diagnosis is achieved
when the characteristic clinical findings are accompanied by a positive ANA test, and often con-
firmed by an antibody directed to Scl-70 by ELISA.
Tables 3–6.1 and 3–6.2 summarize the laboratory evaluation for systemic sclerosis/
scleroderma.
Inflammatory Muscle Diseases Inflammation of the

muscle leading to injury
Description and weakness is the basis
Inflammation of the muscle leading to injury and weakness is the basis of the 3 most common of the 3 most common
but distinct diseases in this category. They are dermatomyositis (DM), polymyositis (PM), and but distinct diseases in
inclusion body myositis. These diseases are more common in women, and their etiology remains this category. They are
unknown, although immune mechanisms have been incriminated. DM may occur as a specific dermatomyositis (DM),
entity or be associated with scleroderma or mixed connective tissue disease. Rarely, it is a man- polymyositis (PM), and
ifestation of a malignancy. Skin manifestations such as a heliotrope rash, the shawl sign, and inclusion body myositis.
Gottron papules are common in DM. Like DM, PM may also be associated with another connec-
tive tissue disease. In addition, it may be associated with viral, parasitic, or bacterial infections.
DM is characterized by immune complex deposition in the vessels and is considered to be in part
a complement-mediated vasculopathy. In contrast, PM appears to reflect direct T-cell-mediated
muscle injury. Inclusion body myositis is a disease of older individuals and is not associated with
malignancy. It is occasionally associated with another connective tissue disease.
TABLE 3–6.1 Laboratory Evaluation for Systemic Sclerosis/Scleroderma

Laboratory Test Scleroderma CREST Syndrome
Pattern of ANA (Hep-2) Speckled Centromeric
Commonly found autoantibody Anti-Scl-70 (greater in diffuse Mostly anticentromeric with a

disease than in localized disease) distinctive pattern on Hep-2 cells
TABLE 3–6.2 Autoantigens and Phenotypes in Systemic Sclerosis/Scleroderma

Autoantigens Description of Phenotype
Scl-70/Topo 1 or 25%-40% of patients with diffuse scleroderma; associated with severe lung disease
topoisomerase 1
ACA or centromere 55%-96% of patients with CREST syndrome. CENP-B (100%) and CENP-C (50%) are the
targets. Seen in Raynaud phenomenon and in about 10% of patients with primary
biliary cirrhosis
RNA polymerase I, II, 4%-20% of patients with diffuse skin disease and renal involvement and less lung and
and III muscle involvement
Fibrillarin (U3 snRNP) 8%-10% of patients with cardiopulmonary and muscle involvement. Higher
prevalence in blacks and Native Americans
PM-Scl A nucleolar complex seen in association with inflammatory muscle disease in

scleroderma
Th/To RNP 10% of patients with limited scleroderma and associated with pulmonary
(endoribonuclease) hypertension and fibrosis
U1 snRNP (U1 RNP Associated with overlap syndrome and mixed connective tissue disease
and polypeptides)
B-23 (nucleophosmin) A nucleolar phosphoprotein associated with pulmonary hypertension and overlap
syndrome
Antisynthetase syndrome, characterized by antisynthetase antibodies that are highly specific

for DM and PM, is seen in about 30% of patients with DM or PM. These patients typically experi-
ence a relatively acute onset of disease, constitutional symptoms such as fever, Raynaud phenom-
enon, arthritis, and interstitial lung diseases. Their hands exhibit a roughening and cracking of the
radial sides of the fingers and the palm, resembling a condition found in people who labor with
their hands such as mechanics, and hence called “mechanic’s hands.” HLA DR 52 has a strong
association (90%) with antisynthetase antibody-positive myositis in people of both European and
African descent. The antisynthetase antibodies include antibodies to aminoacyl-tRNA synthetase;
antihistidyl-tRNA synthetase, also known as Jo-1; anti-signal recognition particle (SRP) antibod-
ies directed against SRP; and anti-Mi-2 antibodies directed against a helicase involved in tran-
scriptional activation.
Diagnosis
Although there are several common features between DM and PM, a characteristic feature of DM
itself is the heliotrope hue around the eyes. Pulmonary interstitial fibrosis is seen in about 10% of
cases in both diseases, occurring in the context of antisynthetase syndromes. There are 5 distinc-
tive features described for both of these diseases. At least 3 of the following features are essential
to fulfill the clinical diagnostic criteria for each:
1. Proximal and symmetrical muscle weakness
2. History of muscle pain and tenderness on palpation
3. Electromyographic evidence of spontaneous muscle activity and myopathic changes
4. Elevated serum or plasma concentrations of muscle enzymes such as aldolase, creatinine
kinase (CK), and AST
5. Muscle biopsy demonstrating cellular inflammation
The laboratory diagnosis begins with documentation of muscle inflammation and injury as
shown by elevation of serum or plasma concentrations of muscle enzymes such as aldolase, CK,
and AST, together with the expected inflammatory histological features on muscle biopsy. The
detection of autoantibodies is found in about one third of the patients, and supports a diagnosis
of inflammatory muscle disease. The antibodies are directed at tRNA synthetases. Anti-Jo-1 is
such an antibody, with specificity to histidyl-tRNA synthetase. It is found in about 40% of patients
with PM, and generally indicates a worse prognosis. It is also more commonly found in patients
TABLE 3–7.1 Laboratory Evaluation for Inflammatory Muscle Diseases

Test Polymyositis Dermatomyositis Inclusion Body Myositis (IBM)
Creatine The CK concentration The CK concentration CK concentrations may be normal or elevated

kinase is elevated >50 times is elevated >50 times no more than 10 times the upper limit of
(CK) and levels reflect and levels reflect normal
disease activity disease activity
Muscle The inflammatory The inflammatory The pattern of inflammation is similar

biopsy infiltrates are usually infiltrate is usually to that seen in polymyositis, with the
within the fascicles around the fascicles. addition of basophilic-rimmed vacuoles
surrounding the The presence within the muscle fiber sarcoplasm that are
healthy muscle fibers; of perifascicular characteristic of IBM; the presence of all 3
no perifascicular atrophy is diagnostic; of the following on muscle biopsy confirms
atrophy; increased complement- IBM and effectively excludes other idiopathic
CD8+ cells mediated inflammatory myopathies: 1) vacuolated
and enhanced vasculopathy is muscle fibers, 2) muscle fiber inclusions
expression of major present with staining characteristics of beta-amyloid
histocompatibility deposits, and 3) demonstration of paired
antigens by muscle helical fibers by electron microscopy or
fibers immunohistological staining
Anti-Jo-1 Present in about 40% Present in about 40% Present in about 40% of patients
antibodies of patients of patients
TABLE 3–7.2 Autoantibodies and Phenotypes in Inflammatory Myositis

Autoantibodies Description of Phenotype
Anti-Jo-1 and other Relatively acute onset of myositis, frequent interstitial lung diseases, fever, Raynaud
antisynthetases phenomenon, mechanic’s hand. Muscle disease dominates the picture in even those
who meet criteria for SLE and RA
Anti-signal recognition Very acute onset of myositis with severe muscle weakness, predominantly in females
particle (SRP) and often in autumn. Rash is absent
Anti-Mi-2 Relatively acute onset of myositis with classical rashes of dermatomyositis such as V
sign and shawl sign
Anti-200/100 Necrotizing myopathy with minimal muscle wasting, preceded by statin therapy, and
very high CPK values
Anti-155/140 Juvenile dermatomyositis and malignancy-associated dermatomyositis
Anti-CADM-140 Clinically amyopathic dermatomyositis with interstitial lung diseases
with pulmonary fibrosis. Jo-1 is more commonly detected in cases of autoimmune myositis than The entity known as
in those with other causes of muscle inflammation. As with many autoimmune diseases, the mixed connective tissue
integration of clinical features with laboratory findings forms the basis of definitive diagnosis. disorder (MCTD) has
Tables 3–7.1 and 3–7.2 present the laboratory evaluation for inflammatory muscle disorders. some of the features of
SLE, some of systemic
sclerosis, and some of
Mixed Connective Tissue Disease polymyositis.
Description
The entity known as mixed connective tissue disease (MCTD) has some of the features of SLE,
some of systemic sclerosis, and some of PM. The patients have variable clinical presentations
with arthralgias, myalgias, fatigue, and Raynaud phenomenon. These features are superim-
posed on other findings that can add in over time, including malar rash, sclerodactyly, arthri-
tis of the hands, and Raynaud phenomenon. Pulmonary manifestations occur in over 85%
of these patients and include interstitial pneumonitis, pulmonary hypertension, progressive
interstitial fibrosis, and, rarely, dysfunction of diaphragm and esophagus. On rare occasion,
patients with MCTD develop diffuse proliferative glomerulonephritis, psychosis, or seizures.
TABLE 3–8 Laboratory Evaluation for Mixed Connective Tissue Disorders

Laboratory Tests Results
Antinuclear antibody Speckled pattern on Hep-2 cells
Autoantibody to extractable nuclear antigens (ENA) Predominantly anti-U1 RNP
Autoantibodies for SLE, SS, and polymyositis Often positive, except for anti-Sm that is negative
The appropriate constellation of clinical findings suggests the need for laboratory testing,
described in Table 3–8.
Diagnosis
The diagnosis of MCTD is largely made on the basis of the clinical features consistent with mul-
tiple autoimmune diseases. It is supported by a high titer of anti-U1 RNP in the serum.
Rheumatoid Arthritis
Description
RA is a systemic RA is a systemic autoimmune connective tissue disorder that primarily affects the synovial
autoimmune connective joints, often starting as a synovitis. It affects 1% to 2% of the adult population worldwide, and
tissue disorder that is predominantly a disease of young women. Susceptibility and resistance to RA is associated
primarily affects the with HLA genotypes. The criteria for diagnosis of RA were revised in 1987 to include clinical
synovial joints, often features, laboratory values, and radiographic findings. To establish a definitive diagnosis, at
starting as a synovitis. least 3 of the following 7 criteria must be present along with morning stiffness for a period of
It affects 1% to 2% of at least 6 weeks:
the adult population
worldwide, and is 1. Arthritis in 3 or more small joints
predominantly a disease 2. Morning stiffness lasting >30 minutes
of young women. 3. Arthritis of the small joints of the hand
4. Rheumatoid nodules
5. Symmetrical arthritis, often with synovitis
6. A positive test for RF
7. Radiographic changes of the affected joints
Diagnosis
An increased serum titer of RF has been a long-standing marker of RA, until the validation of
anti-cyclic citrullinated peptide antibody (anti-CCP). This antibody not only is highly associated
with RA but is also a marker for progressive and erosive joint disease. Anti-CCP is approximately
98% specific and 85% sensitive as a serum marker for RA. RF is an IgM autoantibody directed
against the Fc region of IgG. While high titers of RF are associated with severe RA, it is not specific
for diagnosis of RA, as it is also found in chronic infections and other connective tissue diseases.
Table 3–9 summarizes the laboratory tests useful in the evaluation of RA.
Amyloidosis
Description
Amyloidosis and cryoglobulinemia (which follows) are systemic diseases resulting from the
deposition in the tissues of insoluble proteins from a soluble circulating precursor. Both represent
the consequences of immune dysregulation, and their diagnosis depends on laboratory evalua-
tion and confirmation.
Amyloidosis is a heterogeneous group of diseases resulting from the extracellular deposi-
tion of low-molecular-weight fibrils from a soluble circulating precursor giving a “waxy” or “lar-
daceous” appearance to the infiltrated organs. Ultrastructurally, amyloid deposits are composed
TABLE 3–9 Laboratory Evaluation for Rheumatoid Arthritis

Laboratory Test Results and Significance
Complete blood count (CBC) Patients with RA may have a normochromic, normocytic anemia (Hbg of about
10 g/dL), and elevated platelet count with neutrophilia; in Felty syndrome
there is neutropenia; patients on immunosuppressive therapy have decreased
counts of all lineages
ESR An index of inflammation and often elevated; in RA patients, its level often
parallels disease activity
C-reactive protein This acute phase reactant is increased in RA and is an index of inflammation;
useful in monitoring disease activity over time and in response to therapy
Rheumatoid factor (RF) titer RF is detectable in 70%-80% of patients with RA; diagnostic utility is limited by
its lack of specificity as it is found in almost all patients with cryoglobulinemia,
in 70% of patients with Sjogren syndrome, in 20%-30% of those with SLE, and
in 5%-10% of healthy individuals; its prevalence increases with age
Anti-CCP Most useful, as its specificity is 95%-98% and sensitivity is around 85%;
predicts erosive disease in RA; valuable in diagnosis of early RA; positive titers
to CCP have better predictive value in diagnosis of RA in the IgM-RF-negative
subgroup; negative RF in combination with negative anti-CCP is better in
excluding RA than either alone in patients with polyarthritis
Anti-citrullinated α enolase Predictor of radiographic progression

Anti-citrullinated fibrin Detected in Felty syndrome and vasculitis
Matrix metalloproteinase Radiographic damage

(MMP) 1 and 3
Cartilage oligomeric protein High levels detected in early RA associated with severe disease of both large
(COMP) and small joints
Aggrecan cleavage fragments Noted in slow-onset destructive disease of large and small joints
Pyridinoline cross-links Metabolic marker of activity of bone involvement
Serum cryoglobulins Presence correlates with extra-articular disease
Radiological studies Periarticular osteoporosis, soft tissue swelling, joint space reduction and
erosions should be determined at baseline and monitored with use of
disease-modifying antirheumatic drugs; MRI is sensitive but expensive
Joint fluid analysis If a single joint exhibits heightened inflammation in a patient with
polyarticular disease, need to exclude septic arthritis or crystal-induced
arthritis by cell count and differential, culture, and crystal identification
of unbranching fibrils 8 to 10 nm in width and with a molecular weight of 5 to 25 kd. At least

25 biochemically distinct forms of human amyloid protein have been identified. The 2 most com-
mon forms are primary, with amyloid light chain (AL) derived from light chains of plasma cells, The diagnosis of
and secondary, with amyloid-associated protein (AA), a nonimmunoglobulin protein. Congo red amyloidosis is based
staining of amyloid deposits demonstrates a characteristic apple-green birefringence on polarized on the histological
microscopy, while staining with Thioflavin T produces yellow-green fluorescence. and immunochemical
The classification of amyloidosis is based on whether the amyloidosis is associated with a demonstration of amyloid
plasma cell dyscrasia such as multiple myeloma or light chain myeloma (primary amyloidosis), or deposits in affected
the sequelae of an infectious or inflammatory disease (secondary or reactive amyloidosis). Amy- organs and tissues.
loidosis may also be classified as hereditary or acquired, localized or systemic, or by the type of The preferred tissue for
fibril deposited in tissues, such as transthyretin (TTR) and Alzheimer amyloid precursor protein biopsy is obtained by fine
needle aspiration of the
(APP). A partial list of the chemical classification of human amyloid is given in Table 3–10.1.
abdominal fat pad.
The most common form of the disease, representing 75% to 80% of the cases, is primary
amyloidosis, as an acquired disorder, with multiorgan systemic involvement. Primary amyloido-
sis has a male to female preponderance of 2:1. Its incidence increases with age, often starting at
age 40 years.
Reactive amyloidosis or type AA amyloidosis is a serious outcome of a group of diseases
called autoinflammatory syndromes. This group of diseases represents too much inflammation
TABLE 3–10.1 Chemical Classification of Amyloid

Amyloid Protein Precursor Protein Clinical Syndromes Tissues Involved
AA (Apo) serum AA Chronic inflammation, familial Kidney, liver, and spleen

mediterranean fever (FMF),
familial amyloid nephropathy
(FAN) with urticaria and deafness,
Muckle-Wells syndrome
AL Ig light chain, Primary or myeloma associated Kidney, heart, tongue, bone

kappa or lambda marrow, and peripheral nerves
AH Ig heavy chain Primary or heavy chain disease Kidney, heart, tongue, bone
associated marrow, and peripheral nerves
ATTR Transthyretin FAN, familial amyloidotic Peripheral and autonomic

cardiomyopathy, senile systemic nerves, heart, and kidney
(cardiac) amyloid
AGel Gelsolin Corneal lattice dystrophy and Cornea, cranial and peripheral
cranial neuropathy nerves, kidney
ACys Cystatin C Hereditary cerebral hemorrhage Cranial vessels
with amyloid
Aβ Aβ protein Alzheimer disease, aging Central nervous system

precursor (AβPP)
Atau Tau Alzheimer disease, aging, other Brain

cerebral conditions
Aβ2M β2-Microglobulin Dialysis-related amyloid Synovium, carpal tunnel,

tongue
AApoAI Apolipoprotein A-I Familial amyloidotic Heart, skin, kidney, nerves,

polyneuropathy liver, larynx, and blood vessels
AApoAII Apolipoprotein Familial nephropathy Kidney

A-II
ACal Procalcitonin Medullary thyroid carcinoma Thyroid

AANF Atrial natriuretic Atrial amyloid of aging Cardiac atria
factor
AprP Prion protein Creutzfeldt-Jakob disease, Central nervous system

Gerstmann-Straussler-Scheinker
disease, fatal familial insomnia
secondary to dysregulation of the innate immune system, in the absence of high-titer autoan-
tibodies or antigen-specific T cells. The hereditary autoinflammatory syndromes, also known
as hereditary periodic fever syndromes, represent a group of genetic disorders characterized by
recurrent inflammatory episodes of noninfectious origin, often starting in childhood and persist-
ing lifelong. These syndromes are characterized by a variety of features that include fever, abdomi-
nal symptoms, arthralgias, arthritis, lymphadenopathy, and skin manifestations. An exuberant
acute phase response with elevated C-reactive protein (CRP), serum amyloid A (SAA), and leu-
kocytosis is associated with the inflammatory clinical presentation. The soluble SAA protein is
degraded to the insoluble fibrils composed of AA, which is the hallmark of secondary amyloido-
sis. The mutated genes in these syndromes all code for proteins that play a role in the regulation
of innate immunity.
Diagnosis
The diagnosis of amyloidosis is based on the histological and immunochemical demonstration
of amyloid deposits in affected organs and tissues. The preferred tissue for biopsy is obtained by
fine needle aspiration of the abdominal fat pad. Its advantages over rectal biopsy are that multiple
samples can be obtained for study, and it is less painful and invasive. Since a plasma cell dyscrasia
TABLE 3–10.2 Laboratory Evaluation for Amyloidosis

Laboratory Tests Results/Comments
Abdominal fat pad Preferred site due to ease of obtaining multiple samples, better yield than rectal
aspiration/biopsy biopsy, and less invasive; has replaced rectal biopsy
Labial salivary gland biopsy Better yield than abdominal pad biopsy for both AL and AA amyloidosis
Serum and urine protein Primary amyloidosis is often associated with a monoclonal gammopathy; serum
electrophoresis and urine electrophoresis followed by immunofixation studies to identify the
specific monoclonal protein; assays for serum free light chains and their ratio in
serum are essential to detect light chain disease
Bone marrow biopsy Indicated when serum electrophoresis, serum free light chain assays, and urine
electrophoresis indicate a monoclonal gammopathy; flow cytometry and special
stains for amyloid facilitate diagnosis
Coagulation factor X level About 10% of patients with primary amyloidosis have factor X deficiency; about
half of the patients with isolated and acquired factor X deficiency have primary
amyloidosis; detection of this deficiency prior to biopsy with prothrombin time
and factor X assay is essential
Protein sequencing Useful in identifying genetic abnormalities in hereditary amyloidosis and
identifying rare forms of amyloidosis
Serum amyloid P (SAP) Scintigraphy with radiolabeled SAP used to identify and estimate total body
component scanning burden of amyloid; its value is limited because SAP is obtained from blood
donors and carries potential infectious risk
is commonly found in patients with amyloidosis, a serum protein electrophoresis together with
a determination of serum free kappa and lambda light chains by nephelometry and a calculation
of the kappa/lambda ratio is necessary to exclude a monoclonal gammopathy as the cause of the
amyloidosis. Amyloid fibrils may bind to coagulation factor X causing a coagulopathy. Determi-
nation of the factor X level is important to explain bleeding tendencies in amyloidosis patients
and is useful prior to biopsy of organs and tissues to identify a coagulopathy that would permit
excess bleeding at the biopsy site.
To define the extent of the disease and the type of amyloidosis, the patient should be evalu-
ated for renal, cardiac, pulmonary, neurologic, cutaneous, articular, liver, and spleen involve-
ment. Cardiac involvement is extremely common in primary amyloidosis and much less in
secondary amyloidosis. Virtually all of the familial amyloidosis manifests with nephropathic,
neuropathic, or cardiopathic features. Laboratory evaluation for amyloidosis is summarized in
Table 3–10.2.
Cryoglobulinemia Cryoglobulinemia
Description refers to the presence
in the serum of 1 or
Cryoglobulinemia refers to the presence in the serum of 1 or more immunoglobulins that precipi- more immunoglobulins
tate at a temperature below 37°C. This precipitation is reversible, as it redissolves on warming to that precipitate at a
37°C. The cause of cryoprecipitation remains to be determined. temperature below
Cryoglobulins are classified into 3 types. Type I consists of a single monoclonal immuno- 37°C. This precipitation
globulin that does not have RF activity. It is typically IgM or IgG and less often IgA. Type I, also is reversible, as it
called simple cryoglobulinemia, is often associated with lymphoproliferative malignancies such redissolves on warming
as Waldenstrom macroglobulinemia or multiple myeloma. Patients with this disorder may pres- to 37°C.
ent with features of vasculopathy involving the digits, resulting in gangrene. Type II consists
of monoclonal IgM RF mixed with polyclonal IgG or IgA. The most common association for
this form of cryoglobulinemia is hepatitis C infection. Type II may rarely be associated with
lymphoma. Type III is also a mixed cryoglobulinemia, with polyclonal IgM RF associated with
polyclonal IgG or IgA. Type III is found in patients with connective tissue disease and chronic
infections. Both type II and III cryoglobulinemia patients may show fixation of complement and
be associated with hypocomplementemia. Immune complex vasculitis, arthritis, neuropathy,
TABLE 3–11 Laboratory Evaluation for Cryoglobulinemia

Laboratory Test Comment
Cryocrit This is the (volume of the cryoprecipitate/volume of serum) × 100; necessary to

keep the sample at 37°C until it reaches the laboratory and serum is separated;
serum is then refrigerated at 4°C for 72 h; the cryocrit is then measured after
centrifugation at 4°C; increased fibrinogen and lipids may lead to falsely
elevated values
Immunofixation and Used to evaluate the constituents of the cryoglobulin and their clonality, and
immunodiffusion allow classification as type I, II, or III
Urinalysis, BUN, creatinine To evaluate renal function
C3, C4, and RF To assess for complement fixation by RF in the cryoglobulin
Liver enzymes To evaluate liver function
Hepatitis serology To evaluate hepatitis B or C infections
Renal biopsy and Proteinuria, abnormal urinalysis, and altered renal function are an indication for
immunofluorescence studies renal biopsy with immunofluorescence for renal pathology
Lymph node and bone Indicated when a lymphoproliferative disease is suspected from the type of
marrow biopsy cryoglobulin, usually type I or II
and renal involvement may be the presenting features in patients with type II or III cryoglobu-
linemia.
Diagnosis
When present, the cryoglobulins are quantitated using a Wintrobe tube, and the amount of
cryoglobulin present is reported as a cryocrit. It is important to remember that it is not the
quantity as reported by a cryocrit that is important, but the biological inflammatory properties
of the cryoglobulin. This inflammatory potential is reflected by hypocomplementemia, tissue
inflammation, and organ injury. With therapy, the cryocrit decreases along with mitigation of
inflammatory markers such as CRP, ESR, and complement activation. When a cryoglobulin is
identified, the components comprising the cryoprotein are identified by immunodiffusion and
immunofixation, using specific antisera directed at the immunoglobulin isotypes and against
C3 and C4. Based on the clonality and the constituent isotypes, the cryoglobulin is then cat-
egorized as type I, II, or III. Table 3–11 summarizes the laboratory evaluation for cryoglobu-
linemia.
X-linked DISEASES OF THE IMMUNE SYSTEM

agammaglobulinemia
(XLA) is the prototype X-linked Agammaglobulinemia
humoral immune Description
deficiency. It is a disease
restricted to males, and
X-linked agammaglobulinemia (XLA), also known as Bruton agammaglobulinemia, is the proto-
is characterized by a type humoral immune deficiency. It is a disease restricted to males, and is characterized by a near
near total absence of B total absence of B lymphocytes from an arrest in B lymphocyte development. The deficiency of B
lymphocytes from an cells results in pan-hypogammaglobulinemia. Patients with XLA are asymptomatic for the first
arrest in B lymphocyte several months of life. Recurrent infections manifest between 4 and 12 months after birth as the
development. maternal antibodies wane. Lack of the opsonic antibodies results in recurrent bacterial infections
due to pyogenic, encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influen-
zae, Staphylococcus aureus, and Pseudomonas species. Upper and lower respiratory tract infections
are most common, including otitis media, sinusitis, bronchitis, and pneumonia. Skin infections
and urinary tract infections also occur. The cause of XLA is due to loss of function mutations of
TABLE 3–12 Laboratory Evaluation for X-linked Agammaglobulinemia (XLA)

Laboratory Test Result for XLA Result for Carriers
Serum IgG <200 mg/dL in patients greater than 6 months Normal

of age
Serum IgM and IgA Low to absent if greater than 6 months of age Normal
B-cell markers—CD19 Low at birth and is diagnostic even at <6 months Normal
and CD20 of age
btk gene mutation Although not clinically needed, it is the definitive Definitive test for carrier state
diagnostic test
Antibody response to Markedly decreased to absent Normal

childhood vaccines
a tyrosine kinase protein known as btk that is essential for B-cell development. The availability of
intravenous as well as subcutaneous gammaglobulin replacement therapy has improved outcome
as well as longevity for these patients.
Diagnosis
Early diagnosis is essential to prevent infections and complications of infections, such as bron-
chiectasis, meningitis, bacterial sepsis, septic arthritis, and even osteomyelitis. Recognizing that
B cells are CD19 and CD20 positive, a definitive diagnosis can be made at birth by enumerating
B cells in cord blood by flow cytometry using monoclonal antibodies to CD19 and CD20. In
children less than 6 months of age, measuring serum immunoglobulin concentration is not diag-
nostically useful due to the presence of transplacentally acquired maternal antibody in the blood.
Thus, to establish a diagnosis in the first 6 months, it is necessary to enumerate B cells by flow
cytometry. The molecular diagnosis is made by mutational analysis of the btk gene. This is seldom
needed in clinical practice as clinical features including lack of tonsils and low numbers of CD19
or CD20 cells can establish the diagnosis. Deficient expression of btk protein can be detected by
flow cytometry, a technique that can also be used for carrier detection. Table 3–12 summarizes
the laboratory approach to the diagnosis of this disorder.
Common Variable Immunodeficiency Common variable

Description immunodeficiency
(CVID) affects both
Common variable immunodeficiency (CVID) affects both males and females equally. The pheno- males and females
typic expression of this disease is characterized by hypogammaglobulinemia, and there are many equally. The phenotypic
genetic defects in the B-cell maturation pathway that apparently cause this disorder. The dis- expression of this disease
ease usually manifests in adult life. Unlike agammaglobulinemia, in which B cells and tonsils are is characterized by
absent, patients with CVID have tonsils and normal numbers of B cells in blood and lymphoid tis- hypogammaglobulinemia,
sues. Some patients even have mediastinal and abdominal lymphadenopathy. The primary defect and there are many
in CVID is that the B cells are dysfunctional, and do not differentiate into plasma cells and secrete genetic defects in the
antibody. The clinical presentation is the consequence of the hypogammaglobulinemia, namely, B-cell maturation pathway
recurrent pyogenic infections, often of the upper and lower respiratory tracts. Lack of muco- that apparently cause this
sal immunity also results in enteroviral infections and giardiasis. Autoimmune diseases such as disorder.
immune hemolytic anemia, neutropenia, and pernicious anemia occur. B-cell lymphomas may
manifest with time. Studies of B-cell function in CVID have revealed a subset of CVID patients
who have normal or low normal IgM, IgG, and IgA but fail to make functional antibody to poly-
saccharide and protein antigens. Therapy with intravenous as well as subcutaneous gamma globu-
lin has improved the clinical outcome for CVID patients.
Diagnosis
Table 3–13 describes the laboratory tests useful to establish the clinical diagnosis.
TABLE 3–13 Laboratory Evaluation for Common Variable Immunodeficiency (CVID)

Laboratory Test Result/Comments
Serum protein electrophoresis Marked decrease in the gamma globulin fraction; rarely it may be normal in
the dysfunctional variant
Serum IgM, IgG, and IgA levels Usually low, but may be normal in dysfunctional variant
CD19 and CD20 cells Usually normal, may be increased; rarely, low normal but never absent
CD27+ B cells Absence of this memory B-cell marker is associated with a phenotype that
is more severe and associated with granulomatous pulmonary lesions and
lymphadenopathy
Response to polysaccharide There is a failure to respond to these antigens; the expected 4-fold rise in titer
and protein antigens following vaccination is not observed; defines the functional defect
Hyper-IgM Syndrome
Description
Hyper-IgM syndrome (HIGM) is characterized by markedly reduced IgG and IgA, with normal
to elevated IgM and normal numbers of circulating B cells. The low IgG and IgA is due to an
inability of IgM-positive B cells to switch to other isotypes. The increased IgM reflects polyclonal
expansion of IgM synthesis in response to infections from encapsulated bacteria. Both X-linked
and autosomal recessive forms exist. The molecular causes include the X-linked loss of function
mutation of the CD40 ligand (CD154) found on activated T cells, which is needed to engage with
CD40 on B cells to promote the isotype switch. Other causes include loss of functional mutations
of activation-induced deaminase (AID) and of uracil-DNA glycolase (UNG). These enzymes are
involved in class switch recombination and in mending error-prone repair.
Selective IgA Diagnosis

deficiency is the most The diagnosis is established by measuring serum IgM, IgG, and IgA along with enumerating
common primary CD19 and CD20 cells by flow cytometry. Normal or elevated IgM, with low IgG and IgA, with
immunodeficiency normal B cells, suggests the diagnosis, which can be confirmed by molecular studies.
syndrome. The defect
is due to a B-cell
differentiation arrest in Selective IgA Deficiency
the IgG to IgA isotype
switch.
Description
Selective IgA deficiency is the most common primary immunodeficiency syndrome. Its preva-
lence varies from 1 in 500 in Caucasians to 1 in 10,000 to 15,000 in Asians. The clinical picture is
variable, and includes asymptomatic individuals and those with allergies, autoimmune disorders,
recurrent infections, and gastrointestinal diseases. The defect is due to a B-cell differentiation
arrest in the IgG to IgA isotype switch. There are low numbers of IgA-bearing B cells. Anti-IgA
antibodies are found in the serum of some patients, and these individuals can experience an
anaphylactoid reaction to any blood or blood product containing IgA. These patients are to be
given IgA-deficient blood and blood products and washed red blood cells. Patients with pure IgA
deficiency and normal IgG subclasses should not receive gammaglobulin replacement therapy as
these preparations do not replace IgA and any trace IgA in the gamma globulin preparations can
provoke IgA antibodies and subsequent anaphylactoid reactions.
Diagnosis
The diagnosis is made by documenting an IgA level of <5 mg/dL in the presence of normal IgG
and IgM. It is important to evaluate for IgG subclasses, as subjects with IgA deficiency and a
low IgG2 subclass are prone to recurrent infections. This subset of patients with IgA as well as
low IgG2 subclass does benefit from intravenous or subcutaneous gamma globulin therapy. The
replacement is designed to correct the IgG2 deficiency, as IgG2 is an important opsonin for poly-
saccharide antigens.
DiGeorge Syndrome
Description
DiGeorge syndrome is due to deletion of chromosome 22q11.2 and is part of the spectrum
described as “CATCH 22” (cardiac anomalies, abnormal facies, thymic hypoplasia, cleft pal-
ate, and hypocalcemia). This deletion leads to failure of the development of third and fourth
pharyngeal pouches and consequent abnormalities in the development of the thymus and para-
thyroid glands. Thymic dysfunction leads to T-cell abnormalities, and also B-cell dysfunction,
while parathyroid abnormalities cause hypocalcaemia and tetany. Defects in the third and fourth
pharyngeal pouches also result in congenital heart diseases, anomalies of the great vessels, and
abnormal facies with low-set ears, fish-like mouth, and cleft palate. Patients with complete
DiGeorge syndrome manifest marked defects in T-cell function and are prone to viral infec-
tions. Those with partial DiGeorge syndrome have fewer infections but have cardiac and facial
abnormalities.
Diagnosis
A child with neonatal tetany and abnormal facies should be evaluated for DiGeorge syndrome by
enumerating T and B cells, along with measurement of serum calcium and parathyroid hormone
(PTH). The chromosome 22q11.2 deletion is documented by fluorescence in situ hybridization
(FISH).
Severe Combined Immunodeficiency (SCID) Syndrome SCID syndrome is

characterized by
Description and Diagnosis
profound defects in both
As the name implies, SCID syndrome is characterized by profound defects in both cellular and cellular and humoral
humoral immunity. Affected neonates manifest severe and widespread viral, fungal, and bacte- immunity. Affected
rial infections soon after birth. The protection from maternal antibody is minimal, and the child neonates manifest severe
fails to thrive. Respiratory failure often supervenes and is a cause of death. Patients with this and widespread viral,
disorder were the “bubble babies” decades earlier. Haploidentical, allogeneic bone marrow trans- fungal, and bacterial
plantation has altered the natural history of the disease. The path to recovery is often complicated infections soon after
by graft-versus-host (GVH) disease, and restitution of B-cell function is often incomplete. As a birth.
result, SCID patients may need intravenous gamma globulin to increase their antibody repertoire.
Hematopoietic stem cell transplant with cytokine modulation to facilitate differentiation has been
found to be superior, as there is less GVH disease.
Based on T-cell, B-cell, and NK-cell enumeration, the SCID syndrome is classified accord-
ing to the position of the block/defect in T-cell and B-cell development. Currently, the spec-
trum of primary immunodeficiency diseases includes many underlying mutations. One group
of these mutations results in different combinations of T-, B-, and NK-cell alterations, to pro-
duce SCID and related cellular immunodeficiency diseases (CID). A second group of mutations
alters the amount of antibody to produce primary immunodeficiency diseases. Finally, a third
group of mutations is linked to syndromes involving autoimmunity and immune dysregulation.
Figure 3–2 shows 13 sites where a block in development of a T, B, or NK cell exists. The first 9 are
associated with SCID or CID, and the last 4 are linked to antibody deficiency diseases. This classi-
fication permits an insight to the molecular mechanisms of the disease and provides a framework
for evaluating patients for SCID.
Deficiencies of Complement Proteins

Description
The complement system of proteins and their receptors protect the host against pathogens and
non-self-antigens and also abrogate the emergence of autoimmune diseases by scavenging self-
antigens such as DNA so that they do not become immunogenic. Deficiencies of the comple-
ment system, therefore, result in susceptibility to infections and predispose to autoimmune
diseases such as SLE. Deficiency of C3 results in increased susceptibility to infections by encap-
sulated, pyogenic bacteria. Deficiencies of C5, C6, C7, and C8 result in recurrent or disseminated
8
γδ Thymus γδ
3 P
Myeloid E
progenitor 2 CD 4+ CD 4+
R
4
5
CD 4 + 8+ I
Pro
T αβ P
1 CD 8+ CD 8+
HSC H
6 7
CLP E
9 Pro NK R
NK
Y
Platelets RBC Mature B

Immature B IgD + M+ IgD – M+
IgG + 13
Pro B 10 Pre B 11
12
Denotes site of block. IgA+
FIGURE 3–2 Primary immunodeficiency diseases and associated blocks (shown in numbered
boxes) in T-cell, B-cell, and natural killer (NK)–cell development. CLP, clonal lymphoid progenitor;
HSC, hematopoietic stem cell; NK, natural killer cell.
Neisseria infections. Deficiencies of C1q (which scavenges DNA released from apoptotic cells),
C2, and C4 predispose to SLE and other autoimmune diseases. Deficiency of C1 inhibitor causes
hereditary angioedema (HAE). In type 1 HAE, there is a deficiency of both the antigenic and
functional C1 inhibitor protein. In type 2, the protein is antigenically normal and hence normal
serum levels are noted when it is measured by an antigenic assay. However, in type 2 HAE, the
protein is functionally abnormal and hence cannot inhibit the kinin, complement, kallikrein,
and plasminogen pathways. This results in generation of bradykinin that causes angioedema.
Lack of C1 inhibitor causes C4 consumption even in the basal state so that C4 is always low. In
HAE patients with angioedema, C2 is also decreased. C3 is normal as this activation occurs in
the fluid phase. Acquired C1 INH deficiency leads to a similar clinical phenotype and is called
acquired angioedema (AAE). AAE is seen in lymphoproliferative states such as monoclonal B
cell diseases that activate C1 leading to consumption of C1 INH via C1qrs activation. In autoim-
mune diseases, the autoantibody is directed to C1 INH leading to C1 INH–anti-C1 INH immune
complexes that activate C1qrs. Activated C1 in the fluid phase contributes to the same sequence
of events leading to bradykinin generation and angioedema. The important differentiating fac-
tor is that C1q is normal in HAE, but C1q is low in AAE. Factor I deficiency is associated with
recurrent infections, and factor H deficiency with hemolytic uremic syndrome and age-related
macular degeneration. Deficiency of membrane inhibitors such as decay accelerating factor
(DAF or CD55) and homologous restriction factor (HRF or CD59) causes paroxysmal nocturnal
hemoglobinuria.
Diagnosis
The traditional method to measure the functional integrity of the complement cascade was to
measure the ability of this system to hemolyze antibody-coated sheep red cells in a hemolytic
assay. In this test, the result is reported as titer or the concentration of serum that supports 50%
hemolysis in the S-shaped titration curve (CH50 test). Serum depleted of complement due to
consumption by immune complexes, and serum that is congenitally deficient in complement
proteins both yield low CH50 values. This hemolysis assay has been replaced by enzyme assays
TABLE 3–14 Patterns of Complement Activation

Pattern of Conditions With
Activation CH50 C4 C3 Factor B Activation Pattern
Classical Decreased Decreased Decreased No change SLE, SS, RA, and

cryoglobulinemia
Alternative Decreased No change Decreased Decreased Endotoxemia; type II

MPGN and factor H
mutation
Classical and Decreased Decreased Decreased Decreased SLE, shock, and immune
alternative complex diseases
Fluid phase Decreased Decreased No change No change Hereditary angioedema;

activation— malarial infection
classical (P. vivax)
SLE, systemic lupus erythematosus; SS, Sjogren syndrome; RA, rheumatoid arthritis; MPGN, membranoproliferative
glomerulonephritis.
that detect neoantigens exposed during the activation of terminal complement components.
The hemolysis-based screening test for complement abnormalities in the alternative pathway
is the AH50 assay. In inherited deficiencies of the complement system, specific assays for the
individual complement component must be performed. Further, it must be shown that addition
of that component alone will restore the full hemolytic activity. Table 3–14 provides a profile of
complement activation that is useful in clinical diagnosis. Figure 3–3 provides a framework for
evaluating complement deficiency diseases.
• Recurrent pyogenic infection with normal antibody function

• Disseminated neisserial infection
• Autommune diseases with normal antibody function
• Family history of complement deficiency
Perform CH50
Normal CH50 <5%–10%
Perform AH50 Perform AH50
Normal AH50 <5%–10% Normal AH50 <5%–10%
No complement Suspect factor D, Suspect C1q, C1r, Suspect C3, C5,

deficiency factor B, C1s, C2, C4, or C6, C7, C8, C9,
state or C1 INH deficiency factor H, and/or
properdin factor I deficiency
deficiency
CH50: screening test to check any deficiencies in the complement system

AH50: screening test for complement abnormalities in the alternative pathway
FIGURE 3–3 Evaluation for inherited complement deficiency diseases.

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C H A P T E R
Histocompatibility Testing
and Transplantation
Yash P. Agrawal and Susan L. Saidman 4
LEARNING OBJECTIVES
1. Learn the organization of human leukocyte antigen (HLA) genes and the
molecular details of their gene products.
2. Understand the clinical laboratory tests used to assess histocompatibility.
3. Learn the histocompatibility requirements for the solid organ and stem cell
transplants in clinical use.
CHAPTER OUTLINE
Introduction 83 Kidney 87
HLA Genes and Gene Products 84 Liver 88
Histocompatibility Testing Assays 86 Heart 88
HLA Typing 86 Lung 88
HLA Antibody Screening 86 Pancreas 88
Crossmatching 87 Cornea 88
Histocompatibility Requirements for Solid Hematopoietic Stem Cell
Organ Transplants 87 Transplantation 88
INTRODUCTION
An animal will generally accept an organ transplant from itself, but will reject a transplant from
other animals, even if the donor animal is of the same species. Organ rejection is primarily a
consequence of the interactions between the immune system of the transplant recipient and
the histocompatibility antigens present on the transplanted cells. Clinical laboratories play an
important role in the histocompatibility testing for solid organ as well as hematopoietic stem
cell and bone marrow transplantation (BMT). This chapter provides a brief background to
some of the issues and techniques involved in histocompatibility testing related to transplan-
tation. Other applications of histocompatibility testing such as in the characterization of dis-
ease states (eg, HLA-B27 in ankylosing spondylitis) or before initiation of some drug therapy
(eg, HLA-B∗57:01 is a risk factor for hypersensitivity to abacavir) use similar techniques and are
not discussed further.
The histocompatibility antigens that are the primary stimulus in graft rejection are encoded
by a complex of closely linked genes called the major histocompatibility complex (MHC). In
mice, these genes are located on the H2 region of chromosome 17. In humans, the analogous
MHC region is located in a 4000-kb region on the short arm of chromosome 6 and encodes for
the HLA system (Figure 4–1).
83
84 CHAPTER 4 Histocompatibility Testing and Transplantation
Chromosome 6
Class II Class III Class I
DRB3, Genes
DRB4, or
DPB1 DPA1 DQB1 DQA1 DRB1 DRB5 DRA B C A
FIGURE 4–1 Genes of the human MHC system. The human major histocompatibility complex
(MHC) is located on the short arm of chromosome 6. It contains over 200 genes that can be divided
into different regions (class I-III). Only the major genes encoding the HLA molecules important in
transplantation are shown. The class I region contains genes that encode the α chains of the classic
transplantation antigens, HLA-A, -B, and -C. The class II region has genes that encode both the α and
β chains of HLA-DP, -DQ, and -DR molecules. Genes encoding the α and β chains are designated
as “A” or “B,” and are followed by a number if there is more than 1 gene encoding a particular
chain or a related pseudogene. For example, DRB1 is 1 of the genes that encodes the β chain of
DR molecules. The class III region is located between regions I and II and does not encode for HLA
molecules. (Adapted with permission from Clinical Laboratory Reviews [a newsletter publication of
the Massachusetts General Hospital] 2000;8:3.)
HLA GENES AND GENE PRODUCTS

The HLA class I region encodes for certain glycoprotein molecules that are present on all nucle-
ated cells. The main function of the HLA class I molecules is to bind to fragments resulting from
the breakdown of intracellular pathogens, such as viruses. The HLA molecule and the bound pep-
tide are then presented on the cell surface so that an immune response can be initiated against the
pathogen. Class I molecules consist of 2 noncovalently linked chains. A gene in the MHC encodes
the heavy chain, and a gene outside the MHC on chromosome 15 encodes the β2-microglobulin
In humans, there are light chain. In humans, there are 3 important class I genes known as HLA-A, -B, and -C. These
3 important class I genes genes are highly polymorphic. Polymorphism refers to multiple variations of a single genetic
known as HLA-A, -B, locus and its gene product. Each variation of the gene is called an allele. The HLA-A, -B, and -C
and -C. The class II region genes encode for over 100 gene products that can be defined by serology—that is, by matching
encodes for the α and β antibodies against the different HLA antigens (Table 4–1). However, the antigens identified by
chains that make up the serotyping are far outnumbered by the alleles that are recognized by sequencing the gene. This is
HLA-DR, -DQ, and -DP because not all gene polymorphisms result in distinct antibody specificities, although they may
molecules. stimulate a T-cell immune response.
The class II region encodes for the α and β chains that make up the HLA-DR, -DQ, and -DP
molecules (Figure 4–1). These HLA class II molecules have a more restricted expression than the
class I molecules. They are found mainly on B lymphocytes, dendritic cells, monocytes, activated
T cells, and some endothelial cells. Antigen-presenting cells such as macrophages can phagocytose
and engulf bacteria and other parasites via endocytosis. Once these pathogens gain entry to the cell,
they can be broken down by proteases into peptides that are able to bind to the class II molecules.
These peptides are then presented on the cell surface, and an immune response is initiated. The
class III region contains approximately 40 genes that do not encode HLA molecules, but encode
certain complement components and numerous other proteins not involved in antigen presentation.
The HLA genes are linked together—in what is called a haplotype—on the chromosome and
inherited en bloc. Each individual inherits 1 haplotype from each parent, and the 2 haplotypes
CHAPTER 4 Histocompatibility Testing and Transplantation 85
TABLE 4–1 Number of HLA Alleles and Serologic Specificities

Number of Alleles Determined
HLA Gene by Gene Sequencinga Number of Serologic Specificities
HLA-A >2200 28
HLA-B >2900 70
HLA-C >1700 10
HLA-DRB1 >1300 21
HLA-DQB1 >320 9
a
This number increases continually as new sequences are identified.
represent the HLA genotype. The alleles from both of these haplotypes are expressed on an
individual’s cells. This is referred to as a codominant expression of the gene. Therefore, even though
there are multiple HLA genes, usually only 4 genotypes are possible in the offspring. There is a 25%
chance of any 2 siblings being HLA identical and a 50% chance of the siblings sharing a haplotype.
HLA antigens and alleles are named by the World Health Organization Nomenclature Com-
mittee for Factors of the HLA System. New alleles are named on an ongoing basis as they are
identified, and the number of HLA alleles has grown rapidly, with over 8000 currently listed. The
DNA sequences of all recognized HLA alleles are maintained in the IMGT/HLA Database and
are available online (http://www.ebi.ac.uk/ipd/imgt/hla/). Each HLA allele name follows a strict
format defined by the Nomenclature Committee (Figure 4–2).
Hyphen used to separate Suffix used to denote

gene name from HLA prefix changes in expression
Separator Field separators
HLA-A 02:101:01:02N
HLA prefix Gene Field 4; used to show
differences in a
Field 1; allele group noncoding region
Field 2; specific HLA protein
Field 3; used to show a synonymous DNA

substitution within the coding region
FIGURE 4–2 HLA nomenclature. The letters following the HLA prefix (eg, HLA-A) designate
the gene name in the MHC system. An asterisk (*) separates the gene name from the allele
name. A low-resolution DNA typing is reported at the level of the allele group with only the
first set of digits (eg, HLA-A*02). This is usually the equivalent of a serologic typing result. The
specific HLA protein or allele is reported by the second set of digits in a high-resolution typing
(eg, HLA-A*02:01). The third set of digits is used only when necessary to indicate synonymous
(silent) nucleotide substitutions, and the fourth set of digits is used when necessary to denote
substitutions in noncoding regions of the gene. A letter at the end may be used to denote changes
in expression (eg, N, null or not expressed). (This figure is kindly provided by Professor Steven G.E.
Marsh, Anthony Nolan Research Institute, London, UK, and is from the website hla.alleles.org)
The HLA genes are linked HISTOCOMPATIBILITY TESTING ASSAYS

together—in what is
called a haplotype— HLA Typing
on the chromosome The HLA type of a potential graft recipient or donor can be determined by serology using a
and inherited en bloc. microlymphocytotoxicity assay. T lymphocytes are used for typing class I antigens, and
Each individual inherits
B lymphocytes for typing class II antigens. The assay involves mixing known HLA typing sera with
1 haplotype from
the separated lymphocytes, followed by the addition of complement. Complement-mediated lysis
each parent, and the
2 haplotypes represent
of lymphocytes follows when the antibody in the serum binds to the appropriate HLA antigen.
the HLA genotype. The extent of cell lysis is visualized under a fluorescence microscope after exposing the cells to
DNA-binding dyes such as ethidium bromide. HLA typing for class I and II antigens typically
involves mixing the cells with over 200 different HLA typing sera, each in a different well of a
microtiter tray.
Molecular methods allow high-resolution HLA typing at the allele level, so different alleles
that cannot be distinguished by serologic assays can be identified. Such techniques also have
applications in typing nonlymphocytes (eg, blasts or epithelial cells from cheek swabs) and in
patients with cytopenias. One technique involves amplification of genomic DNA by polymerase
chain reaction (PCR) (see Chapter 2) using primers that are locus specific (eg, all DQB1 alleles)
or group specific (eg, all DR4 alleles). The amplified DNA is then hybridized with a panel of
sequence-specific oligonucleotide probes (PCR-SSOP) specific for each allele or group of alleles.
Even a single nucleotide mismatch will prevent the annealing of the probe. The bound probe is
visualized using various methods, including autoradiography and color development, by blotting
the DNA onto multiple membranes (dot blot hybridization). More commonly, the probes may
be bound to a single membrane (reverse hybridization) or to groups of different colored beads
(Luminex technology).
In a related technique, sequence-specific primers for an allele are used in a PCR amplification
reaction (PCR-SSP). The presence or absence of PCR amplification is detected by gel electro-
phoresis and ethidium bromide visualization. In this technique, a positive amplification reaction
signifies the presence of that specific allele.
In recent times, the PCR-SSOP and PCR-SSP methods are being replaced by sequence-based
typing (SBT) methods using PCR combined with the Sanger method of DNA sequencing. The
SBT methods do not always resolve sequence ambiguities that may be important in BMT, espe-
cially when the ambiguities are due to the cis/trans assignment of nucleotide bases in alleles of
heterozygous individuals. Next-generation sequencing (NGS) holds the promise of resolving
these ambiguities because the technique utilizes the creation of DNA libraries to produce clones
of phase-defined sequences that are then sequenced in a massively parallel manner. The technique
allows the simultaneous sequencing of multiple long DNA fragments in a single run.
HLA Antibody Screening

The patient’s serum can be screened for the presence of antibodies that may have resulted from
prior transfusions, pregnancies, or transplants. Serum from patients is screened for reactivity
against a panel of lymphocytes or purified HLA molecules from individuals (panel cells) with
known HLA types. Screening can be done using either cell-based or solid-phase assays. In the
cell-based assays, patient serum is mixed with different panel cells. If the cells are lysed (in a
cytotoxicity assay) or if antibody binds to them (detected using a flow cytometry assay), then it
is evident that the patient’s serum contains HLA antibody against the antigens expressed on that
panel cell. In the solid-phase assays, HLA antigens are extracted from the panel cells. The antigens
are purified and bound to a solid support, either to the wells of an ELISA plate or to colored
beads that can be detected using flow cytometry or Luminex technology. Antibody binding to
the molecules on the well or bead is detected using an enzyme or fluorescence conjugated anti-
immunoglobulin reagent.
The number of panel cells showing lysis or antibody binding is noted, and the results are
expressed as percent panel reactive antibody (PRA). Patients with a high PRA are referred to as
“sensitized.” If the patient’s serum reacts with 90% of the panel cells, it is likely that the patient will
have to wait longer for a compatible donor than someone who shows a PRA of 0% (ie, no HLA
antibody). Identification of the HLA antigen specificity of the antibody is an important feature of
the antibody screening assays, and may reduce unnecessary crossmatches between patients who
have antibodies against specific HLA antigens and donors who are positive for those antigens.
Knowledge of antibody specificity can also increase opportunities for identifying compatible donors
for these difficult-to-match patients. Highly sensitized (high PRA) patients may also be managed
differently posttransplant since they have a higher risk of rejection. Pretransplant screening for
HLA antibodies is also indicated in patients undergoing autologous stem cell transplants, since
posttransplant patients with a high PRA are more likely to become refractory to platelet transfusions
and may require careful observation regarding their need for HLA-matched platelets.
Crossmatching
The lymphocyte crossmatch is a critical step, especially before renal transplantation. It is
also important in sensitized patients who are undergoing a heart or lung transplant. In this
assay, the graft recipient’s serum is mixed with donor lymphocytes (similar to that shown for
red blood cells in Chapter 2 under Blood Crossmatch). If the recipient has preformed HLA
antibodies against donor antigens, the cells will be lysed, the crossmatch is considered positive,
and the transplant will likely not be done. The test may be performed using a variation of the
microlymphocytotoxicity assay that is used for HLA typing. In this test, antihuman globulin
(AHG) is added to the microtiter wells after mixing the serum with the lymphocytes, which
greatly increases the sensitivity of the assay. The crossmatch may also be performed by measuring
antibody binding to cells by flow cytometry.
HISTOCOMPATIBILITY REQUIREMENTS FOR SOLID

ORGAN TRANSPLANTS
In general, HLA matching is not an absolute requirement with respect to solid organ transplants In general, HLA matching
(Table 4–2). It is usually a requirement for BMT. The application of HLA typing, crossmatching, is not an absolute
and antibody screening in histocompatibility testing prior to transplantation of specific organs or requirement with respect
tissues is described hereafter. to solid organ transplants.
It is usually a requirement
for bone marrow
Kidney transplantation.
Renal transplantation from living donors, whether HLA matched or unmatched, is preferable
to kidney transplantation from deceased donors. The half-life of a transplanted deceased donor
kidney is about 8 years, as compared with 12 and 26 years for kidneys matched at 1 or 2 haplotypes
from living donors. In the United States, nearly 35% of the kidney transplants are from living
donors, including genetically unrelated donors such as spouses or friends. In a large multicenter
study involving primary cadaveric renal transplants, multiple factors were shown to influence
the outcome. These included the age of donor and recipient, presence of diabetes in the recipient,
TABLE 4–2 General Requirements for HLA and ABO Blood Group Matching
in Transplants
Organ HLA ABO
a
Kidney No Yes
Cornea No No
Liver No Yes
Heart No Yes
Lung No Yes
a
Pancreas No Yes
Stem cell/bone marrow Yes No

a
HLA matching preferable but not required.
the cause of the donor’s death, cold ischemic time of the donated kidney prior to transplant,
In the United States, the transplant center, and matching for HLA antigens. The advantages of HLA-matched
nearly 35% of the kidney transplants include a reduced need for antirejection treatment and posttransplant dialysis, as
transplants are from well as significantly improved long-term survival. Other important predictors of outcome are
living donors, including ABO blood group compatibility, PRA, and crossmatch between donor lymphocytes and recipient
genetically unrelated serum. A positive crossmatch is usually a contraindication to transplant. However, techniques to
donors such as spouses or remove recipient circulating HLA and/or ABO antibody have been recently developed, allowing
friends. successful transplantation with previously incompatible kidneys.
Liver
HLA matching does not appear to correlate with better outcomes in liver transplantation. There
are conflicting reports regarding the importance of a negative crossmatch in the pretransplant
setting, but donor-specific HLA antibodies are not a contraindication to transplantation. ABO
matching, in contrast, is associated with better outcomes in both adult and pediatric populations.
Heart
The benefits of HLA matching in heart transplantation are difficult to evaluate because there are
few studies that utilize prospective HLA matching. The usual priorities in cardiac transplantation
are short ischemia time for the donated heart prior to transplant (<4 hours), heart size, and blood
group matching. Most centers screen for HLA antibodies prior to transplant surgery and perform,
when possible, a prospective crossmatch using donor cells only on sensitized patients. The
presence of HLA-specific antibodies and a positive crossmatch against donor cells are generally
accepted to be associated with an adverse outcome.
Lung
While there may be a small survival advantage with HLA-matched lung transplants, there is no
consensus on the hierarchical importance of the various HLA loci. Lungs are allocated on the
basis of ABO compatibility and size. Similar to heart transplant patients, crossmatches are usually
done only on patients known to have HLA antibody.
Pancreas
Pancreata are transplanted based on ABO compatibility. HLA antibody screening is performed on
transplant candidates, and a positive crossmatch is usually a contraindication to transplantation
with that donor. Patients with pancreas-only transplantation have a lower survival rate than those
undergoing combined pancreas and kidney transplantation. Most pancreata are transplanted
in patients undergoing kidney transplants for diabetic renal failure. Evidence suggests that the
kidney–pancreas transplant combination is associated with a long-term reduction in mortality, as
compared with renal transplant only, in end-stage diabetic renal disease.
Cornea
The cornea is the most commonly transplanted tissue. There is no convincing evidence for
the utility of HLA and ABO group matching for corneal transplants. However, the long-term
graft survival is approximately 50% and rejection remains the most common cause of graft loss.
Because corneal rejection is not life-threatening, the routine use of systemic immunosuppression
for prevention of rejection is not a consideration.
HEMATOPOIETIC STEM CELL TRANSPLANTATION

Hematopoietic bone marrow/stem cell transplantation is a therapeutic option in which
normal hematopoietic stem cells are used to replace abnormal hematopoietic stem cells or
to reconstitute the bone marrow of patients undergoing high-dose cytotoxic therapy for
malignancy. The hematopoietic stem and progenitor cells can be harvested from the bone
marrow under general anesthesia or from the peripheral blood after giving the donor multiple
doses of growth factors/cytokines such as granulocyte colony-stimulating factor (G-CSF) or
granulocyte–monocyte colony-stimulating factor (GM-CSF).
HLA matching is usually a requirement for allogeneic stem cell transplantation. A positive Hematopoietic bone
crossmatch between donor and recipient is a strong predictor of graft failure, but because most marrow/stem cell
patients and donors are HLA matched, this is rarely a concern. Recently, success has been obtained transplantation is a
in cases involving partially mismatched BMT, which has increased transplantation opportunities therapeutic option
for many patients. in which normal
In the past, serologic techniques were used extensively for HLA typing in BMT. As noted hematopoietic stem
earlier, it is now known that the number of identifiable serologic specificities at any locus is far cells are used to replace
less than the number of true alleles at that locus (Table 4–1). Individuals who appear to be HLA abnormal hematopoietic
stem cells or to
matched after serologic typing may in fact have some mismatched alleles. Patients transplanted
reconstitute the bone
with genotypically HLA-matched marrow from siblings or unrelated individuals have a graft
marrow of patients
failure rate of approximately 2%. The risk of graft failure is much higher in the presence of a
undergoing high-dose
mismatch of 2 or more class I alleles. cytotoxic therapy for
The widespread availability of high-resolution allelic typing methods that allow better malignancy.
matching of donors with recipients has resulted in improved outcomes.
A substantial portion of this chapter also appeared previously in Clinical Laboratory Reviews
(a newsletter publication of the Massachusetts General Hospital, 2000;8:3).
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C H A P T E R
Infectious Diseases
Eric D. Spitzer 5
LEARNING OBJECTIVES
1. Determine if an organism of interest is a bacterium, fungus, parasite, or virus
and learn how it is further classified among related organisms.
2. Learn the organisms that produce the commonly encountered and better
characterized infectious diseases.
3. Distinguish pathogenic organisms from those found in normal flora.
4. Learn the laboratory test results associated with the individual infectious
diseases and how they are used in establishing the diagnosis.
CHAPTER OUTLINE
Introduction 92 Bone Infections/Osteomyelitis 112
Laboratory Tests for Infectious Agents 96 Infections of the Joints 113
Direct Stains 96 Infections of the Skin and Adjacent
Culture 96 Soft Tissue 115
Identification of Organisms From Acute Bacterial Infections 115
Positive Cultures 96 Lyme Disease 118
Susceptibility Testing 97 Cat-scratch Disease and Bacillary
Antigen Detection 97 Angiomatosis 120
Nucleic Acid Amplification 97 Fungal Infections 120
Serology 98 Viral Infections With Prominent
Sepsis and Bloodstream Infections 98 Skin Manifestations 121
Bacteremia 98 Varicella Zoster Viral Infection 121
Infections Caused by Rickettsia, Measles (Rubeola) and Rubella 123
Ehrlichia, and Related Organisms 100 Eye Infections 124
Fungemia 101 Infections of the Larynx, Pharynx,
Parasitic Infections of the Blood 102 Mouth, Ear, Orbit, and Sinuses 125
Malaria 102 Infections of the Lung and
Babesiosis 102 Pleurae 127
Viral Infections of the Blood 103 Tuberculosis 130
Infectious Mononucleosis/ Legionella Infections 131
Epstein–Barr Virus 104 Nocardiosis 133
Cytomegalovirus 104 Pneumocystis jirovecii Pneumonia 134
Parvovirus B19 105 Dimorphic Fungi and Other
Endocarditis: Infection of the Heart 106 Fungal Infections 135
Infections of the Central Nervous Respiratory Virus Infections 137
System 107 Infections of the Gastrointestinal
Acute Bacterial Meningitis 107 Tract 137
Acute Viral Meningitis 110 Viruses Inducing Gastroenteritis 137
Chronic Meningitis 111 Aerobic Bacterial Infections 138
Encephalitis 111 Clostridium difficile Infections 138
Brain Abscess 112 Protozoal Infections 140
91
92 CHAPTER 5 Infectious Diseases
Intestinal Helminth Infections of the Female Genital

Infections 141 Tract 148
Food Poisoning 145 Sexually Transmitted Diseases 149
Botulism 145 Syphilis 149
Pyelonephritis and Urinary Gonorrhea 150
Tract Infections 146 Chlamydial Infections 152
Infections of the Male Genital Herpes Simplex Virus
Tract 148 Infections 153
INTRODUCTION
Humans live in a world of microbes. Many types of microbes are part of the normal human
flora and rarely cause disease. Others have a greater potential for virulence and can cause disease
depending on complex interactions between the host and the microbe. A small group of organ-
isms are highly virulent and usually cause disease whenever they infect humans. This chapter
on infectious diseases and clinical microbiology focuses on common pathogens and frequently
encountered clinical syndromes. Infectious agents include a daunting array of viruses, bacteria,
fungi, protozoans, and helminths. Table 5–1 provides information on the basic microbiology and
clinical significance of common pathogens. The organisms are grouped based on shared proper-
ties because these are often relevant to the diagnostic process.
Because of the large number of potential pathogens, it is not technically possible, practical,
or cost-effective to attempt to rule out all of them in each patient who may have an infection.
It is, therefore important for the clinician to know what organisms are most likely in a particular
patient and whether routine diagnostic tests will detect them or whether specialized tests are
needed. Identification of the causative agent is usually important for determining the most appro-
priate therapy. It can also have infection control or public health implications. The clinical find-
ings are the first major clues in determining the site of infection and identifying a pathogenic
organism. For example, a cough is usually indicative of a process in the respiratory tract while
pain on urination is a clue to a urinary tract infection (UTI). Radiographic studies can further
clarify the type of process and may point to specific categories of organisms.
Often, a single species of microbe can cause multiple syndromes. Conversely, a single syn-
drome may be caused by multiple organisms. This can lead to a potentially vast array of diagnos-
tic possibilities. In order to determine the diagnosis in a timely and cost-efficient manner, it is
essential to take into consideration the clinical setting. For example, the organisms responsible for
a community-acquired pneumonia are usually different from those that cause nosocomial pneu-
monia. If the patient is immunosuppressed, this further enlarges the list of potential organisms.
The organization of It also matters whether the immunosuppression is due to decreased cell-mediated immunity, for
this chapter reflects the example, due to HIV infection or inhibitors of tumor necrosis factor (TNF), versus neutropenia
common associations secondary to chemotherapy because each has its own associated group of opportunistic infec-
between selected tions. Other underlying conditions such as diabetes or sickle cell disease, or the presence of pros-
organisms and the site thetic devices, are associated with specific infections. A history of travel or exposure to arthropod
of infection. vectors may raise the possibility of additional organisms.
The organization of this chapter reflects the common associations between selected organ-
isms and the site of infection. The discussion of a particular organism in a specific anatomic
site in this chapter does not imply that infection by that organism is restricted to that loca-
tion. Several infectious diseases, such as viral hepatitis (see Chapter 16) and Helicobacter pylori
infections (see Chapter 15), are presented elsewhere in this book because these infections are
intimately associated with a specific organ or tissue. The organisms and diseases selected for
presentation in this chapter were chosen primarily by their incidence, with a preference for
common infections. Many lower-incidence infections also have been included because they
are often within a differential diagnosis, new information on their diagnosis and treatment is
emerging, or they can be overlooked if appropriate tests are not requested. The chapter focuses
on infections commonly encountered in the United States, although several travel-associated
infections are discussed.
A general clinical approach to the patient with an infectious disease is diagrammed in
Figure 5–1.
CHAPTER 5 Infectious Diseases 93
TABLE 5–1 Selected Clinically Significant Microorganisms

Aerobic gram-positive cocci Aerobic gram-negative bacilli Mycoplasma and Ureaplasma
Occur singly or in pairs, tetrads, chains, or clusters Enterobacteriaceae; rod-like organisms; Small highly pleomorphic organisms;
Catalase positive oxidase-negative; ferment sugars difficult to observe with routine stains
Micrococcus and require complex medium for growth
Citrobacter
Staphylococcus aureus Edwardsiella Mycoplasma
Coagulase-negative staphylococci Enterobacter Ureaplasma
Catalase negative Escherichia Treponemes
Aerococcus Ewingella
Enterococcus faecalis Hafnia Spiral organisms that may or may not stain with
routine stains and require complex medium
Enterococcus faecium Klebsiella
or animal host for growth
Gemella Morganella morganii
Borrelia
Leuconostoc Proteus
Leptospira
Streptococcus agalactiae (group B streptococci) Providencia
Spirillum
Streptococcus gallolyticus (bovis) (group D Salmonella
nonenterococcus) Treponema
Serratia
Streptococcus dysgalactiae (multiple species Shigella Anaerobic gram-negative bacilli
within group C or group G streptococci are Yersinia enterocolitica
classified as S. dysgalactiae) Bacteroides
Yersinia pestis
Streptococcus anginosus/intermedius group Bilophila
Yersinia pseudotuberculosis Fusobacterium
(viridans streptococci)
Streptococcus mitis (viridans streptococci) Aerobic gram-negative bacilli Prevotella
Streptococcus mutans group (viridans Porphyromonas
Nonenterobacteriaceae, fermentative; rod-like
streptococci)
organisms; oxidase-positive; ferment sugars Anaerobic gram-negative cocci
Streptococcus pneumoniae (pneumococcus)
Streptococcus pyogenes (group A streptococci) Aeromonas Acidaminococcus
Streptococcus salivarius group (viridans Chromobacterium Veillonella
streptococci) Pasteurella
Anaerobic gram-positive bacilli:
Streptococcus sanguis group (viridans Plesiomonas
nonspore forming
streptococci) Vibrio
Actinomyces
Aerobic gram-negative cocci Aerobic gram-negative bacilli Lactobacillus
Occur singly or in pairs or clumps; catalase Nonenterobacteriaceae; rod-like organisms; Propionibacterium
positive, oxidase positive catalase positive; do not ferment sugars;
Anaerobic gram-positive bacilli: spore forming
oxidase variable
Moraxella catarrhalis (Branhamella catarrhalis)
Acinetobacter Clostridium
Neisseria gonorrhoeae
Neisseria meningiditis Alcaligenes Anaerobic gram-positive cocci
Burkholderia
Aerobic gram-positive bacilli Finegoldia
Chryseobacterium (Flavobacterium)
Parvimonas
Rod-like organisms; only Bacillus species Empedobacter (Flavobacterium)
produce spores; some organisms in category Peptostreptococcus
Flaviomonas
are partially acid-fast Staphylococcus saccharolyticus
Flavobacterium
Bacillus Pseudomonas Obligate intracellular bacteria
Corynebacterium Stenotrophomonas
Chlamydia
Erysipelothrix
Aerobic fastidious gram-negative bacilli Coxiella
Gardnerella vaginalis (Gram variable) Ehrlichia
Lactobacillus Small, straight or curved gram-negative
Rickettsia
Listeria bacilli or coccobacilli; may require special
Nocardia
conditions for adequate growth Fungi of medical significancea
Aggregatibacter
Mycobacteria Acremonium Malassezia
Afipia
Aspergillus Microsporum
Rod-like organisms; acid-fast stain positive; some Bartonella
Bordetella Blastomyces Mucor
stains are gram-positive; most are slow growing
Brucella Candida Paracoccidioides
Mycobacterium tuberculosis Campylobacter (microaerophilic) Coccidioides Penicillium
Mycobacterium avium complex Cardiobacterium Cryptococcus Pseudallescheria (Scedosporium)
Mycobacterium kansasii Eikenella Epidermophyton Rhizopus
Mycobacterium marinum Francisella Fonsecaea Sporothrix
Mycobacterium fortuitum complex (rapid Haemophilus
grower) Fusarium Trichophyton
Helicobacter pylori
Mycobacterium abscessus (rapid grower) Kingella Geotrichum Trichosporon
Mycobacterium chelonae (rapid grower) Legionella Histoplasma Wangiella

TABLE 5–1 Selected Clinically Significant Microorganisms (continued)

Virusesb
Family Representative Species Pathogenic for Humans Family Representative Species Pathogenic for Humans
DNA viruses Arenaviridae Lymphocytic choriomeningitis virus

Poxviridae Vaccinia virus Lassa fever virus
Variola virus (smallpox) Junin (Argentine hemorrhagic
Molluscum contagiosum virus fever virus)
Herpesviridae Herpes simplex virus, type 1 Machupo (Bolivian hemorrhagic
Herpes simplex virus, type 2 fever virus)
Varicella zoster virus Picornaviridae Enteroviruses
Epstein–Barr virus Poliovirus (3 types)
Cytomegalovirus Coxsackie A virus (23 types)
Human herpesvirus 6 (HHV 6) Coxsackie B virus (6 types)
Human herpesvirus 8 (HHV 8, Kaposi Echovirus (30 types)
sarcoma-associated herpesvirus) Enteroviruses 68-71 (4)
Adenoviridae Human adenoviruses (51 serotypes) Rhinovirus (common cold virus)
Papillomaviridae Human papillomaviruses (>96 types) (>115 types)
Polyomaviridae BK virus Hepatitis A virus (enterovirus 72)
JC virus Caliciviridae Noroviruses
Parvoviridae B19 virus (human parvovirus) Norwalk and Norwalk-like gastroenteritis
viruses
Hepadnaviridae Hepatitis B virus
Hepeviridae Hepatitis E virus (enterically transmitted)
RNA viruses Astroviridae Human astroviruses
Reoviridae Orthoreoviruses Coronaviridae Human coronaviruses
Colorado tick fever virus SARS-CoV
Rotaviruses A-C Flaviviridae Flaviviruses (mosquito-borne)
Paramyxoviridae Respiroviruses St. Louis and Japanese encephalitis
Parainfluenza virus, types 1 and 3 viruses, West Nile virus, yellow fever virus,
Morbilliviruses dengue virus
Measles virus Flaviviruses (tick-borne)
Rubulaviruses Omsk hemorrhagic fever, European
Mumps virus, parainfluenza virus, types 2 and 4 and Far Eastern tick-borne encephalitis
viruses
Pneumoviruses
Hepatitis C virus (parenterally transmitted)
Respiratory syncytial virus
Togaviridae Alphaviruses
Metapneumovirus
Western, Eastern, and Venezuelan
Rhabdoviridae Rabies virus
equine encephalitis viruses; Ross
Filoviride Marburg and Ebola viruses River and Semliki Forest viruses
Orthomyxoviridae Influenza A virus (mosquito-borne)
Influenza B virus Rubivirus
Bunyaviridae Orthobunyaviruses (mosquito-transmitted) Rubella virus
California serogroup (eg, California encephalitis and Retroviridae Human T-cell lymphotropic virus
La Crosse viruses) HTLV 1 and 2
Hantaviruses (rodent-associated) Human immunodeficiency virus
Hantaan virus (hemorrhagic fever with renal syndrome), HIV 1 and 2
Sin Nombre virus (hantavirus pulmonary syndrome)
Subviral agents Endolimax Flagellates (blood, tissue)

Iodamoeba Leishmania
Satellites
Blastocystis Trypanosoma
Hepatitis delta (D) virus
Prions Amebas (other body sites) Flagellates (other body sites)
Kuru, Creutzfeldt–Jakob disease (CJD), Naegleria Trichomonas vaginalis
Gerstmann–Straussler–Scheinker syndrome Acanthamoeba Ciliates (intestinal)
(GSS), fatal familial insomnia (FFI) Balamuthia
Balantidium
Parasites of clinical significancec Flagellates (intestinal)
Coccidia (intestinal)
Giardia
Protozoa Cryptosporidium
Dientamoeba
Amebas (intestinal) Cyclospora
Trichomonas hominis
Entamoeba Isospora

TABLE 5–1 Selected Clinically Significant Microorganisms (continued)

Coccidia (other body sites) Trichostrongylus Hymenolepis
Toxoplasma Trichuris Taenia solium
Taenia saginata
Microsporidia (intestinal) (Tissue)
Enterocytozoon Trichinella (Tissue—larval forms)
Encephalitozoon Visceral larva migrans (Toxocara canis T. solium
or Toxocara cati) Echinococcus
Microsporidia (other body sites)
Ocular larva migrans (T. canis or T. cati)
Encephalitozoon Trematodes (flukes)
Cutaneous larva migrans (Ancylostoma
Enterocytozoon braziliense or Ancylostoma caninum) (Intestinal)
Dracunculus Fasciolopsis
Sporozoa (blood, tissue)
Angiostrongylus Echinostoma
Plasmodium
Gnathostoma Metagonimus
Babesia
Anisakis (Liver/lung)
Fungal-like organism (formerly
Capillaria Clonorchis
classified as a protozoan)
Pneumocystis (Blood and tissues) Opisthorchis
Wuchereria Fasciola
Nematodes (roundworms) Paragonimus
Brugia
(Intestinal)
Loa (Blood and tissue)
Ascaris
Onchocerca Schistosoma mansoni (intestine)
Enterobius
Ancylostoma Cestodes (tapeworms) Schistosoma japonicum (intestine)
Necator (Intestinal) Schistosoma haematobium (bladder)
Strongyloides Diphyllobothrium
a
The fungi are listed by genus. As with the bacteria, certain species within a genus are more commonly associated with infections than other species. The list includes the fungi
associated with the majority of human fungal infections. There are many more fungi in nature than are listed here. Compiled from data in McGinnis MR, Rinaldi MG. Some
medically important fungi and their common synonyms and obsolete names. Clin Infect Dis. 1997;25:15.
b
The viruses are grouped by family. Listed within each family are representative viral species that are pathogenic for humans. Compiled from data in Miller MJ. Viral taxonomy.
Clin Infect Dis. 1997;25:18–20 and updated based on Knipe DM, Howley PM, eds. Fields’ Virology. 5th ed. Philadelphia, PA: Wolters Kluwer; 2007.
c
These organisms are listed by type of organism and most common site of infection. They are listed by genus only unless an individual species within a genus is located in 1
category and another species within the same genus is in a different category. Compiled from data in Garcia LS. Classification of human parasites. Clin Infect Dis. 1997;25:21.
List clinical signs and symptoms to identify

Collect appropriate samples for analysis
specific organs and tissues likely
(see Chapter 2) from site of infection.
to be infected.
Order appropriate tests to detect the most likely infectious agents (see Tables 5–3 to 5–42).
This may include Gram stain and bacterial culture for common infections and/or specialized
tests such as fungal culture, nucleic acid amplification tests for viral agents, microscopy for
parasites, or serologic tests, depending on the differential diagnosis.
Begin empiric treatment if necessary with agents effective against most likely pathogens.
The laboratory isolates/identifies pathogenic organism(s). If more than 1 organism is detected

in the sample, determine the pathogen(s) based on clinical correlation and knowledge
of normal flora.
The laboratory performs tests for antibiotic susceptibilities of pathogenic organisms.

When these results become available, modify treatment if necessary to target the pathogenic
organism(s). Susceptibility tests are widely used for bacterial infections but are less frequently
used for fungal, viral, and parasitic infections.
FIGURE 5–1 A general clinical approach to the patient with an infectious disease.
LABORATORY TESTS FOR INFECTIOUS AGENTS

The laboratory diagnosis of infectious disease utilizes 5 distinct types of tests: direct examination,
culture, antigen detection, nucleic acid detection, and serology. Each of these tests has strengths
and weaknesses. The types of test(s) that are used in a specific case depend in large part on the
organisms that are in the differential diagnosis, as well as the type of specimens that are available.
See Chapter 2 for illustrations of these laboratory methods.
Direct Stains
Direct examination involves preparing a smear of the specimen and then using an appropriate
staining technique to detect the relevant microorganisms. The Gram stain is rapid and detects
most types of bacterial pathogens if they are present in sufficient numbers. It provides a prelimi-
nary characterization in terms of the Gram reaction, that is, positive or negative, as well as the
morphology (cocci, coccobacilli, or bacilli) and arrangement of the cells (individual cells, pairs,
chains, or clusters). It also provides information on the host response, for example, the presence
or absence of neutrophils. This stain is routinely performed on most specimen types including
respiratory specimens, sterile fluids, tissue biopsies, wounds, and abscesses. It is not routinely
performed on stool because of the large numbers of normal flora, urine because similar infor-
mation is available from the urinalysis, and blood because of the small number of organisms
typically found in cases of bacteremia. The analytic sensitivity of the Gram stain is relatively low
The laboratory diagnosis because the observation of an average of 1 organism per oil immersion field corresponds to a
of infectious disease concentration of 105 organisms per milliliter in the specimen. The sensitivity can be increased by
utilizes 5 distinct types of concentration of the specimen through centrifugation. Other direct stains include acid-fast stains
tests: direct examination, for mycobacteria and calcofluor white for fungi. Wright stains of peripheral blood are used to
culture, antigen detect Plasmodium and Babesia infections. Routine hematoxylin and eosin stains as well as Gram,
detection, nucleic acid acid-fast bacteria (AFB), and Gomori methenamine silver (GMS) stains can reveal the presence
detection, and serology. of microorganisms in paraffin-embedded tissue in the surgical pathology laboratory.
Culture
Isolation of organisms in pure culture continues to be the mainstay of microbiologic diagnosis.
In general, culture is very sensitive and specific and remains the “gold standard” for diagnosing
many types of infection. Culture provides relatively rapid detection (within usually 1-3 days) of
a wide array of organisms that are then available for definitive identification and antimicrobial
susceptibility testing. One of the great advantages of culture assays is that microbiology laborato-
ries routinely inoculate a combination of nonselective and selective media that will support the
growth of many types of pathogenic bacteria. The person ordering the test does not need to spec-
ify which organism or organisms are suspected of causing the infection. A clinician who submits
a blood-culture bottle does not have to order a Staphylococcus culture, a Streptococcus culture, a
gram-negative rod culture, etc. Nonetheless, it is essential to remember that routine cultures only
detect typical bacteria. If other classes of agents, such as mycobacteria, fungi, parasites, or viruses,
are in the differential diagnosis, then the clinician must request the appropriate tests. For example,
mycobacteria and fungal cultures utilize media designed to inhibit the growth of routine bacteria
and they also require prolonged incubation.
Identification of Organisms From Positive Cultures

The majority of bacterial pathogens are routinely identified by the presence of specific combina-
tions of phenotypic traits including production of enzymes, such as catalase, coagulase, oxidase,
and urease, ability to ferment or utilize different types of sugars, and additional metabolic reac-
tions. Many of these individual tests have been combined into commercial identification kits. In
contrast, slow-growing organisms, such as mycobacteria, and bacteria that are difficult to identify
with traditional biochemical methods are now often identified by nucleic acid amplification and
sequencing of conserved genes such as 16S ribosomal RNA or RNA polymerase. Yeasts are gener-
ally identified with biochemical tests similar to those used for bacteria. Finally, identification of
molds is based on colonial and microscopic morphology.
More recently, commercial identification systems have been developed that use matrix-
assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) to
generate protein spectra that can be compared with databases to identify bacteria and other
microbes. The advantage of MALDI-TOF/MS is that it requires few reagents and can identify
organisms in minutes as opposed to the 12- to 24-hour incubations required for most biochemi-
cal panels.
Susceptibility Testing
Once an organism has been identified in the clinical microbiology laboratory, the second major
task, in most cases, is determining the organism’s antibiotic susceptibility profile. Susceptibility
tests involve the measurement of the minimum inhibitory concentration (MIC), the lowest con-
centration of antimicrobial agent that inhibits the growth of the organism. The reference method
is the microbroth dilution technique. The MICs are then interpreted as sensitive, intermediate, or
resistant according to tables of interpretive breakpoints that are related to therapeutically achiev-
able serum levels for each antibiotic. Other susceptibility methods such as disk diffusion (mea-
suring the diameter of the zone of inhibition surrounding an antibiotic-containing disk) can be
correlated with the results of the broth dilution technique.
Susceptibility testing has become increasingly complex due to the widespread dissemina-
tion of increasingly resistant pathogens that include methicillin-resistant Staphylococcus aureus
(MRSA); vancomycin-resistant enterococci (VRE); and multidrug-resistant strains of Klebsiella
pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. These organisms harbor a
variety of resistance mechanisms that include altered penicillin-binding proteins, extended-spec-
trum beta-lactamases, carbapenemases, inducible clindamycin resistance, and multidrug efflux
pumps. While molecular methods can be used for rapid detection of specific resistance genes,
such as the mecA gene in MRSA and the vanA and vanB genes in VRE, most susceptibility testing
depends on phenotypic (MIC-based) methods that require overnight incubation.
Antigen Detection
Antigen detection tests do not require the growth of microorganisms. Therefore, they have the
potential to provide rapid detection of infectious agents. Immunoassays that detect soluble anti-
gens vary in speed, complexity, sensitivity, and specificity. Usually there is a trade-off between
simplicity and sensitivity. Immunochromatographic assays require very few procedural steps
and are the basis of many rapid point-of-care tests. These assays are less sensitive than tradi-
tional solid-phase enzyme immunoassays (EIAs) that are used for batch testing in the laboratory.
Unlike culture-based assays, an immunoassay can only detect the organism that binds to the
reagent antibodies; a separate assay must be performed for each organism. Immunoassays gener-
ally exhibit good but not perfect specificity. Furthermore, they cannot distinguish viable from
nonviable organisms. Direct or indirect fluorescent immunoassays utilize microscopy to identify
organisms that bind the reagent antibody. These assays often have high sensitivity and specificity
compared with other types of immunoassays, but they require extensive training to insure proper
interpretation.
Nucleic Acid Amplification

The introduction of nucleic acid amplification techniques (NAATs) has revolutionized several
areas of infectious disease testing including HIV viral load testing, diagnosis of Herpes simplex
virus (HSV) encephalitis, and rapid detection of MRSA. They are particularly valuable for detect-
ing difficult-to-grow or slow-growing organisms that may be present in small numbers. NAAT
tests have the potential for very high sensitivity and specificity. However, as with antigen tests,
they can only be used to diagnose infections caused by organisms that the assay detects. Current
technology is not at the stage where it can eliminate the use of traditional culture methods in the
routine bacteriology laboratory. Nonetheless, this is a rapidly changing field as illustrated by the
widespread adoption of multiplex NAAT assays that can simultaneously detect a panel of com-
mon respiratory viruses.
Serology
Serologic tests detect host antibodies that are produced in response to infection with a particular
infectious agent. The most important limitation of these assays is that antibody is usually not
detectable early in the course of infection. Even if antibody is detected, it may represent a past
infection. Serologic diagnosis usually requires demonstration of seroconversion, a 4-fold rise in
IgG titer between sequential specimens, or a positive IgM assay. While the latter is usually thought
of as a marker of acute infection, IgM can persist for 6 to 12 months. For the reasons outlined
above, serologic assays are mainly used to diagnose infections that cannot be detected using more
direct methods.
SEPSIS AND BLOODSTREAM INFECTIONS

Bacteremia
Description
Normally the blood is sterile. Infection in any of the organs or tissues can result in entry of
bacteria into the circulation. Replication of bacteria in the blood can contribute to the signs
and symptoms of sepsis (eg, fever, tachycardia, leukocytosis, and hypotension) and may lead to
dissemination of the organism to other tissues and organs; however, patients can be septic with-
out having demonstrable bacteria in their blood. Bacteremia is often described as transient,
intermittent, or continuous based on the number of positive specimens. Transient bacteremia
occurs when small numbers of a commensal organism present on a mucosal surface gain access
to the bloodstream. These infections are usually not clinically significant when they occur in
an otherwise healthy host. Intermittent bacteremias are usually associated with a sequestered
infection somewhere in the organs or tissues (eg, an abscess). Continuous bacteremias are asso-
ciated with an intravascular focus of infection. Examples include endocarditis or an infected
vascular catheter.
Diagnosis
Blood cultures are routinely collected as part of the diagnostic evaluation of patients who
present with signs and symptoms of sepsis or disseminated infection. To maximize sensitivity
and specificity, it is recommended that 2 to 3 sets of blood cultures be collected per septic epi-
sode. Most hospitals use continuous blood culture systems that utilize colorimetric, fluores-
cent, or manometric methods to defect bacterial growth. Positive bottles are then subcultured
to agar plates for further evaluation of the organisms.
To identify intermittent bacteremias, the timing of the blood collection is important to maxi-
mize the likelihood of finding organisms while they are in the blood (see the section on sample
collection in microbiology in Chapter 2). Ideally, blood from patients with intermittent bactere-
mias is collected during the hour before a temperature spike, but this is not practical because the
febrile episodes are often not predictable. It is common practice for blood to be collected at 30- to
60-minute intervals (if possible) when a febrile patient is suspected of having an intermittent bac-
teremia. As one might expect, one is much more likely to detect a continuous bacteremia in the
first blood culture than to detect an intermittent bacteremia.
A major problem in the A major problem in the interpretation of the blood culture results is incidental contami-
interpretation of the nation of the specimen with the normal bacterial flora from the skin (Table 5–2). The clinical
blood culture results is significance of a positive blood culture is dependent on both the number of positive specimens
incidental contamination and the type of organism. The isolation of recognized pathogens such as S. aureus, Streptococ-
of the specimen with the cus pneumoniae, beta-hemolytic streptococci (Streptococcus pyogenes and Streptococcus agalac-
normal bacterial flora tiae), enterococci, gram-negative rods (aerobic and anaerobic), or yeast from 1 or more blood
from the skin. cultures is almost always clinically significant (Table 5–3). In contrast, if only 1 of the multiple
blood culture specimens is positive for an organism found on the skin (eg, coagulase-negative
staphylococci or Corynebacterium spp.), the result is likely to reflect contamination during
specimen collection rather than true septicemia. This frequently encountered problem is 1 rea-
son why at least 2 blood samples should be collected for blood cultures. Although skin-derived
TABLE 5–2 Normal Body Floraa

Mouth Vagina Sputum
More common More common Often contaminated from

Anaerobic streptococci Bacteroides upper respiratory tract; most
Aerobic streptococci (not group A) Veillonella commonly Staphylococcus aureus,
Staphylococcus epidermidis Anaerobic streptococci S. epidermidis, Haemophilus spp.,
Lactobacillus Aerobic streptococci (groups B Corynebacterium, Streptococcus viridans,
Streptococcus pneumoniae and D) Enterobacteriaceae, Candida spp.,
Moraxella catarrhalis Lactobacillus Neisseria spp.
Bacteroides S. epidermidis
Veillonella Staphylococcus aureus Jejunum
Nonpathogenic Neisseria Corynebacterium Lactobacillus
Candida Gardnerella vaginalis
Enterococcus
Less common Candida
Streptococcus pyogenes (group A) Less common Terminal ileum and colon
Neisseria meningitides Listeria monocytogenes (95% are anaerobes)
Staphylococcus aureus Ureaplasma
Haemophilus influenzae, Enterobacteriaceae Neisseria More common
Actinomyces Clostridium perfringens Peptococcus
Actinomyces Peptostreptococcus
Throat (nasopharynx and oropharynx) Clostridium
Skin Bacteroides
More common Fusobacterium
Streptococcus More common Enterobacteriaceae
Nonpathogenic Neisseria S. epidermidis and other coagulase-negative Lactobacillus
Staphylococcus aureus staphylococci Enterococcus spp.
Corynebacterium Staphylococcus aureus
Less common
H. influenzae Corynebacterium
Pseudomonas
Less common Malassezia
Staphylococcus aureus
S. pyogenes (group A) Less common S. epidermidis
N. meningitides Streptococcus (group A) Actinomyces
Enterobacteriaceae Bacillus Nontuberculous mycobacteria
Bacteroides Peptostreptococcus Treponema
Candida Nontuberculous mycobacteria Candida
Candida Clostridium difficile
Nose
Eye Bacillus subtilis
More common
Corynebacterium More common Sterile areas (selected)
S. epidermidis S. epidermidis Bronchi
Aerobic Streptococcus (nongroup A) Haemophilus
Blood
Streptococcus aureus Corynebacterium
Haemophilus spp. Cerebrospinal fluid
Uncommon
Less common S. pyogenes Joint fluid
S. pneumoniae Moraxella Pleural fluid
H. influenzae Neisseria Pericardial fluid
N. meningitides S. pneumoniae Peritoneal fluid
S. pyogenes Enterobacteriaceae
a
Listed by genus only if species is not specified (eg, Bacteroides) or by genus and species (eg, Haemophilus influenzae) if specific organism is known.
Modified from Ravel R. Clinical Laboratory Medicine. 6th ed. St. Louis, MO: Mosby-Year Book; 1995:666–667.
bacteria are often thought of as nonpathogenic, it is important to remember that they can cause
clinically significant infections, particularly in immunosuppressed patients and in patients with
intravascular catheters or prosthetic devices. The isolation of the same skin flora organism in 2
separately collected specimens increases the probability that it represents a clinically significant To avoid the problem
bacteremia. of contamination of the
To avoid the problem of contamination of the blood culture bottles with skin organisms, blood culture bottles
meticulous preparation of the skin with a bactericidal agent is necessary. The number of blood with skin organisms,
cultures required for detection of a pathogenic organism is determined by the volume of blood meticulous preparation
collected per bottle, the timing of the blood collection, the type of organism producing the infec- of the skin with a
tion, and previous antibiotic exposure. Three or more blood culture collections may be required bactericidal agent
to document the presence of certain organisms. is necessary.
TABLE 5–3 Clinical Significance of Organisms That Are Frequently

Isolated From Blood Cultures
Organism Probability That the Organism Is a True Pathogena,b
Gram-positive aerobic bacteria

Staphylococcus aureus High
Coagulase-negative staphylococci Low/intermediate
Enterococcus spp. Intermediate/high
Viridans group streptococci Intermediate
Βeta-hemolytic streptococci High
Streptococcus pneumoniae High
Bacillus spp. Low
Corynebacterium spp. Low
Gram-negative aerobic bacteria

Escherichia coli High
Klebsiella pneumoniae High
Enterobacter cloacae High
Pseudomonas aeruginosa High
Acinetobacter baumanii Intermediate/high
Anaerobic bacteria
Clostridium spp. Intermediate
Propionibacterium spp. Low
Bacteroides fragilis group High
Yeast
Candida spp. High
Cryptococcus neoformans High
High, 90% to 100%; intermediate, >10% to <90%; low, 0% to 10%.
a
b
Based on data from Pien BC, et al. The clinical and prognostic importance of positive blood cultures in adults. Am J Med.
2010;123:819–828 and Weinstein MP, et al. The clinical significance of positive blood cultures in the 1990s: a prospective
comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis.
1997;24;584–602.
Infections Caused by Rickettsia, Ehrlichia, and Related Organisms

Description
Rickettsia, Ehrlichia, and Anaplasma are obligate intracellular bacteria that cannot be detected in
routine bacterial cultures. These organisms are transmitted to humans by ticks; therefore, the risk
of acquiring these infections depends on the geographic distribution of the tick vectors and the
time of year (see Table 5–4).
Rickettsia rickettsii, the agent of Rocky Mountain spotted fever (RMSF), is mainly transmit-
ted by the dog tick (Dermacentor variabilis) and infects endothelial cells. The resulting vascular
injury elicits a widespread vasculitis, consisting of vasodilation with perivascular edema, and at
times complicated by thrombosis and hemorrhage. Erythrocytes extravasating into the dermis
form nonblanching petechial or purpuric lesions. The characteristic rash is often absent during
the early stages of infection, and the infection can progress to a life-threatening encephalitis if not
promptly treated.
Ehrlichia chafeensis is transmitted by the lone star tick (Amblyomma americanum) and
infects monocytes. Patients present with nonspecific findings including fever, leukopenia,
thrombocytopenia, and/or elevations of hepatic enzymes. Similar clinical manifestations are seen
with Anaplasma (formerly Ehrlichia) phagocytophilum that is transmitted by deer ticks (Ixodes
spp.) and infects granulocytes.
Diagnosis
None of these agents can be cultured on artificial media. RMSF is usually diagnosed retrospec-
tively with serologic tests; however, this should not delay treatment that should be initiated based
on clinical findings and potential history of exposure. If the rash is present, organisms can be
TABLE 5–4 Evaluation for Rickettsial Diseases and Selected Disorders Caused by Related Organisms
Mechanism of
Disease Etiologic Agent Transmission to Humans Clinical Features Laboratory Tests
Rocky Rickettsia rickettsii Tick vector Higher rate seasonally and in specific Serology: increase in
Mountain geographic areas; rash from a vasculitis, antibody titer after exposure;
spotted fever fever, and headache immunohistochemical tests
give rapid result, but sensitivity
is low at about 70%
Boutonneuse Rickettsia conorii Tick vector Seasonal: distribution in Europe, Asia, Same as for Rocky Mountain
fever and Africa; rash, fever, headache, and spotted fever
black spot at the site of tick attachment
Rickettsial pox Rickettsia akari Bite of mouse mite Similar to but milder than Rocky Serology: increase in antibody
Mountain spotted fever and titer after exposure
Boutonneuse fever; uncommon
in the United States
Murine Rickettsia typhi Flea feces inoculated into Seasonal and geographic; rash, fever, Serology: increase in antibody
typhus flea bite wound on human and headache titer after exposure
Epidemic Rickettsia Infected louse feces Associated with domestic crowding plus Serology: increase in antibody
typhus prowazekii inoculated into human poor hygiene (as in refugee camps); titer after exposure
skin wounds clinically similar to Rocky Mountain
spotted fever
Scrub typhus Orientia Bite of a larval mite Rash, fever, and headache Serology: increase in antibody
tsutsugamushi titer after exposure
Q fever Coxiella burnetii Inhalation of infected Acutely, it is usually an asymptomatic Serology: increase in antibody
aerosols, ingestion of or self-limited febrile pneumonia; can titer after exposure
contaminated dairy become chronic with damage to heart
products, or, rarely, valves and bone
by tick vector
Erhlichiosis Ehrlichia Tick vector From asymptomatic to a severe Rocky Serology: increase in antibody
chaffeensis Mountain spotted fever-like illness titer after exposure; PCR
Anaplasmosis Anaplasma Tick vector Fever, headache, myalgias Serology: increase in antibody
phagocytophilium titer after exposure; PCR
demonstrated by immunohistochemical staining of a skin biopsy in 70% of cases. Examination of

peripheral blood smears in patients with Anaplasma can reveal the presence of organisms within
inclusions in neutrophils, but many cases are negative. Ehrlichia infects monocytes but is rarely
observed in peripheral blood smears. Polymerase chain reaction (PCR) of blood and/or serologic
tests are the best methods for diagnosing Anaplasma and Ehrlichia infections.
Fungemia
Description
Yeast belonging to the genus Candida are a major cause of hospital-acquired bloodstream infec-
tions. These organisms are frequently part of the oral and gastrointestinal flora. Treatment with
broad-spectrum antibiotics that disrupt the normal bacterial flora, the presence of intravenous
catheters, and neutropenia all predispose to the development of candidemia.
Cryptococcus neoformans and Histoplasma capsulatum are important causes of fungemia in
patients with markedly depressed cell-mediated immunity (Cryptococcus is further discussed in
the section “Chronic Meningitis” and Histoplasma is further discussed in the section “Infections
of the Lung and Pleurae”). Although molds such as Aspergillus spp. can cause disseminated infec-
tions in immunosuppressed patients, they are rarely detected in the bloodstream.
Diagnosis
Candidemia can usually be detected with routine blood cultures. Specialized techniques
(eg, lysis–centrifugation cultures) are usually required to detect H. capsulatum and may
Malaria is 1 of the largest enhance the detection of C. neoformans. Immunoassays that detect antigens produced by
causes of mortality and H. capsulatum and C. neoformans are also used to diagnose disseminated infections caused
morbidity in the world. by these organisms.
Individuals who travel
to areas where malaria
is endemic and develop Parasitic Infections of the Blood
fever within weeks
Overview
of return should be
suspected of suffering Several vector-borne parasites can infect the blood. These include protozoans such as Plasmo-
from malaria. dium spp. (malaria), Babesia spp., and Trypanosoma spp., and nematodes such as the agents of
lymphatic filariasis. Plasmodium infections are an important cause of nonspecific febrile illnesses
in returning travelers, and Babesia infections are endemic in the United States; these infections
are discussed below. Other blood parasites are uncommon in the United States.
Malaria
Description
Malaria is one of the largest causes of mortality and morbidity in the world. Individuals who
travel to areas where malaria is endemic and develop fever within weeks of return should be sus-
pected of suffering from malaria.
There are 4 species of Plasmodium that cause most cases of human malaria. These parasites
are transmitted by Anopheles mosquitoes that are widely distributed throughout Africa, Asia, and
Latin America. The most dangerous of the 4 species is Plasmodium falciparum. This organism can
achieve very high levels of parasitemia and adheres to capillary endothelium, and this can lead
to severe organ damage. P. falciparum infection may be fatal within days. P. vivax and P. ovale
are morphologically similar and generally cause less severe infection, but unlike P. falciparum,
they can establish persistent infection and cause relapses several months after the initial infec-
tion. P. malariae is the least virulent species and can cause low-level infection that may cause few
symptoms, but it can persist for years. P. knowlesi, which infects monkeys, can also cause human
infections.
Diagnosis
Currently, the diagnosis of malaria and the identification of each of the 4 species of Plasmodium
responsible for malaria are based on the microscopic examination of stained erythrocytes in thick
and thin blood films. These organisms have maturation cycles involving a variety of specific struc-
tures in the RBC, including ring trophozoites, growing trophozoites, mature schizonts, and game-
tocytes. The stippling of the RBCs with different dot patterns is also important in differentiating
between the 4 species. Thus, the size and shape of the various malarial forms, their alteration of
RBC morphology, and the stippling pattern in the RBCs provide the identification of the particu-
lar type of malaria. Quantitation of the level of parasitemia is also important. Marked parasitemia
is a poor prognostic sign for P. falciparum infection. However, ill patients may have relatively low
levels of parasitemia due to trapping of organisms in capillaries. PCR is starting to be used for the
diagnosis of malaria and is particularly useful when low levels of parasitemia make it difficult to
identify individual species. A rapid immunodiagnostic test for malaria may also be useful in set-
tings where microscopy is not immediately available. Table 5–5 summarizes the relevant labora-
tory information for the diagnosis of malaria.
Babesia are protozoa that, Babesiosis

like Plasmodium species, Description
infect erythrocytes.
They are delivered to
Babesia species are protozoa that, like Plasmodium species, infect erythrocytes. They are delivered
the infected host by the to the infected host by the same tick (Ixodes) that transmits the agent of Lyme disease and human
same tick (Ixodes) that granulocytic anaplasmosis. Babesiosis mimics malaria in that it causes hemolysis, fever, anorexia,
transmits the agent of and hemoglobinuria. In the United States, B. microti is responsible for most cases of human babe-
Lyme disease. siosis. In Europe, B. bovis and B. bigemina have been implicated as agents of human disease.
Babesiosis occurs mainly in the Northeast and upper Midwest in the United States. It affects
TABLE 5–5 Laboratory Evaluation for Malaria

Laboratory Test Results/Comments
Identification of The first-line test in the diagnosis of malaria; for best results, blood should be collected
organisms within from the patient during or after a febrile episode and before the administration of
RBC in blood smears antiparasitic medications; to rule out malaria, it is recommended that negative results
be demonstrated in blood samples collected every 6-8 h for 24 h
Preparation of smears: thick and thin blood smears should be prepared; thick smears
allow for a rapid examination of a relatively large volume of blood and have
approximately a 10-fold increase in sensitivity over thin smears; thin smears allow for
superior preservation of morphology and are needed for species determination; the
best stain is the aqueous Giemsa stain buffered with phosphate to pH 7.2
Reading of smears: no single criterion except for the crescentiform (banana-shaped)
gametocyte of Plasmodium falciparum is pathognomonic; multiple morphologic
criteria are used to make the diagnosis of malaria and determine speciation of the
Plasmodium; it is very difficult to speciate when mixed Plasmodium infections occur;
PCR can be used to confirm identification when morphology is not definitive
Quantitation of Reported as percent of RBC parasitized or as number of parasites per 100 WBC;
parasitemia quantitation should be repeated after treatment to monitor effectiveness
patients of all ages, but most cases occur during the sixth and seventh decades of life. The infec-
tion from Babesia tends to be self-limited. In most cases, it lasts from weeks to months, following
an incubation period of 1 to 6 weeks. Mild symptoms, including malaise, fever, and headache,
characterize the disease in normal hosts, but asplenic patients often develop severe infections
with high levels of parasitemia.
Diagnosis
The laboratory diagnosis rests upon the identification of the Babesia organisms inside erythrocytes
in stained thick and thin peripheral blood smears. There are a number of morphologic features
that differentiate Babesia from Plasmodium. Despite its relative shortcomings, serologic testing
for B. microti can be performed as noted in Table 5–6. The level of parasitemia with Babesia does
not always correlate with the severity of symptoms.
Viral Infections of the Blood

Overview
Many viruses such as varicella zoster virus (VZV), measles, enteroviruses, and arboviruses (such
as West Nile virus) exhibit a viremic phase. These viruses also exhibit organ-specific manifesta-
tions and are discussed in other sections of this chapter. Cytomegalovirus (CMV), Epstein–Barr
virus (EBV), and parvovirus B19 are discussed below because they are common viruses that can
have a direct effect on the blood.
TABLE 5–6 Laboratory Evaluation for Babesia

Identification of organism Primary method of diagnosis; differentiating features in infected RBC

in RBC on blood film suggesting Babesia rather than Plasmodium include 1) a tetrad of structures
that resembles a “Maltese cross” and 2) the absence of pigment granules
Indirect immunofluorescent A titer of >1:64 is considered indicative of exposure to the organisms, and
testing for antibodies to a titer of >1:256 is diagnostic for an acute Babesia infection; at titers <1:256,
B. microti the result does not clearly differentiate between patients who were exposed
in the past and those who are actively infected
PCR amplification Useful for confirming identification in low-level infections or detecting very
low numbers of organisms; not routinely available
Infectious Mononucleosis/Epstein–Barr Virus

Description
Most cases of mononucleosis are caused by EBV, a member of the herpesvirus family that
The diagnosis of EBV- infects B lymphocytes and causes them to proliferate. This in turn stimulates the proliferation of
associated infectious cytotoxic T cells that control the active infection but do not eradicate the latent state. Infection
mononucleosis is usually with EBV is extremely common, and most individuals have asymptomatic infections. Patients
confirmed by a positive with infectious mononucleosis typically present with fever, sore throat, and enlarged cervical
serum heterophile lymph nodes.
antibody test that In addition to mononucleosis, EBV is associated with 2 types of human tumor (Burkitt lym-
detects the presence phoma and nasopharyngeal carcinoma) and is responsible for lymphoproliferative disorders in
of antibodies that patients with severe immunosuppression following organ transplantation or AIDS. It also causes
agglutinate horse or cow oral hairy leukoplakia in HIV-infected patients.
erythrocytes.
Diagnosis
Large atypical lymphocytes (cytotoxic T cells) are usually present in peripheral blood smears of
patients with infectious mononucleosis caused by EBV, but they are also found in many other
infections. The diagnosis of EBV-associated infectious mononucleosis is usually confirmed by a
positive serum heterophile antibody test that detects the presence of antibodies that agglutinate
horse or cow erythrocytes. The heterophile test is often negative in young children or in patients
with atypical presentations; EBV-specific serologic tests are especially important in establishing
the diagnosis in these situations. Quantitation of EBV DNA in peripheral blood is important in
the diagnosis and management of EBV-associated lymphoproliferative disease in solid organ and
bone marrow transplant recipients.
The use of laboratory tests in the diagnosis of infectious mononucleosis is shown in Table 5–7.
Cytomegalovirus
Description
CMV causes several clinical syndromes. It infects leukocytes, where it remains latent in immuno-
competent individuals but readily reactivates in immunosuppressed individuals. CMV is a leading
cause of opportunistic infections in transplant recipients and AIDS patients. In transplant recipi-
ents, it often presents as a nonspecific febrile illness, but it can also cause more invasive infections
including esophagitis, hepatitis, colitis, pneumonitis, and retinitis, particularly in severely immu-
nocompromised patients. CMV is also the most common congenital viral infection. It affects
TABLE 5–7 Evaluation for Infectious Mononucleosis

Laboratory Tests Comments
Heterophile Heterophile antibodies are IgM antibodies reactive with antigens on the cells from
antibody tests multiple species, and on this basis are termed heterophilic; they are detected in
agglutination assays with horse or sheep RBCs or by their capacity to induce an
agglutination response on antigen-coated latex particles; in infectious mononucleosis,
the heterophile antibody test result is positive approximately 1 week after the
symptoms first appear; the highest titers appear in the second to third week of
the illness; relatively high titers persist for up to 8 weeks
Antibodies to EBV- Although heterophile-negative infectious mononucleosis is uncommon, it does

specific antigens occur, mostly in young children; in these cases, a characteristic clinical picture and
the presence of IgM against the EBV viral capsid antigen (VCA-IgM) or a rising titer
of VCA-IgG can confirm acute infection, whereas a negative VCA-IgM and positive
VCA-IgG and positive EBNA antibodies (EBV nuclear antigen) indicate past infection
WBC differential Patients with mononucleosis typically have a mild-to-moderate leukocytosis after
the first week, with more than 60% of the WBCs as lymphocytes and 10%-20% of
all lymphocytes being atypical; the maximum percentage of atypical lymphocytes
appears between days 5 and 10 after the onset of symptoms, with a decrease to
normal by approximately 3 weeks in most patients
EBV, Epstein–Barr virus.
approximately 40,000 infants born each year in the United States. Hematogenous spread appears
to be responsible for transmission of the virus to the fetus. Most congenital infections occur when
the mother has a primary CMV infection during the pregnancy. Neonates can also acquire the CMV is the most common
infection from maternal breast milk. Approximately 10% of infants congenitally infected with congenital viral infection.
CMV are symptomatic at birth. Common sites of involvement are liver, spleen, lungs, and cen- It affects approximately
tral nervous system (CNS). Because specific antiviral therapy is available for treatment of these 40,000 infants born each
infants, rapid detection of CMV infection is necessary. Although most congenitally infected year in the United States.
infants are asymptomatic at birth, approximately 10% to 15% of these will develop later problems
such as hearing loss and other neurologic problems. In children and young adults, primary CMV
infection can cause a mononucleosis-like illness.
Diagnosis
The detection of CMV in blood and tissues generally correlates with active disease, whereas detec-
tion of CMV in urine is not necessarily diagnostic of active CMV disease, even in immunocom-
promised patients. Quantitative PCR assays of CMV DNA in blood correlate with the likelihood
of severe infection. Congenital CMV infection is established when CMV is isolated from the urine
of neonates less than 3 weeks of age. CMV isolation from respiratory secretions of bone marrow
transplant recipients is likely to be clinically significant because interstitial pneumonia is a compli-
cation of bone marrow transplantation. In AIDS patients, shedding of CMV in respiratory secre-
tions does not always correlate with active infection. CMV serology is used to determine whether
donors and/or recipients are latently infected with CMV. This has important implications for pre-
venting subsequent infections. Diagnostic testing for CMV is summarized in Table 5–8.
Parvovirus B19
Description
Parvovirus B19 is a small single-stranded DNA virus that is transmitted by respiratory droplets.
It is a common cause of infection in children in whom it causes a distinctive rash known as
fifth disease. In adults, it often causes a significant arthropathy. An unusual feature of this virus
is that it replicates in erythroid precursor cells and causes a temporary cessation of RBC pro-
duction until the virus is cleared by the immune system. In normal hosts this has little, if any,
TABLE 5–8 Laboratory Testing for Cytomegalovirus (CMV)

Laboratory Test Result/Comment
Conventional cell culture Detection of CMV infection typically requires 7-28 days with conventional viral
inoculation of cell cultures; CMV can be isolated from a variety of specimens
including blood, bronchoalveolar lavage fluid, urine, and tissue
Shell vial assay This is a modification of the conventional cell culture methodology for
more rapid viral detection; viruses are detected earlier using this technique
than conventional cell culture because the specimen is inoculated onto the
monolayer of cultured cells by low-speed centrifugation; this enhances the
infectivity of the cultured cells; this assay can often provide a positive result
in 1-2 days
CMV antigen testing A test is available for the identification of CMV antigen in polymorphonuclear
leukocytes using a monoclonal antibody directed at a specific CMV protein;
the assay is semiquantitative and permits monitoring response to therapy
Enzyme immunoassay (EIA) This test is used to detect antibodies to CMV; seroconversion from negative
to positive or a significant rise in anti-CMV IgG titer provides evidence of
infection; assays for anti-CMV IgM are available, but detection of anti-CMV IgM
does not always indicate primary infection because the IgM can persist for up
to 18 months; most adults are seropositive and therefore serologic tests have
limited utility for diagnosis
Polymerase chain reaction DNA- and RNA-based detection is available for the detection of CMV in
(PCR)-based detection of CMV peripheral blood leukocytes and tissues; quantitative assays are important
for diagnosis of active infection in immunosuppressed patients
consequence, but in patients who have a chronic hemolytic anemia such as sickle cell disease or
hereditary spherocytosis, the parvovirus B19 infection causes a transient aplastic crisis in which
there is a profound drop in the hematocrit. Parvovirus B19 can cause a chronic anemia in immu-
The clinical features of nocompromised patients who are unable to clear the virus. Intrauterine infection of the fetus can
infectious endocarditis, a also cause a severe anemia that leads to congestive failure and hydrops fetalis.
microbial infection of the
valvular or nonvalvular
endothelium of the heart, Diagnosis
depend on the type Acute parvovirus infection can be confirmed by demonstration of IgM antibodies or detec-
of organism, location, tion of viral DNA by PCR. During a transient aplastic crisis, the reticulocyte count decreases to
and type of valve. <0.1% even as the hematocrit is declining.
ENDOCARDITIS: INFECTION OF THE HEART

Description
The clinical features of infectious endocarditis, a microbial infection of the valvular or nonvalvu-
lar endothelium of the heart, depend on the type of organism, location, and type of valve. Acute
infective endocarditis can present with temperatures ≥103°F, shaking chills, rapid worsening of
valve function, and a variety of septic embolic complications. Subacute bacterial endocarditis is
characterized by a low-grade or absent fever (as a result of infection by low-virulence organisms),
and a variety of nonspecific signs and symptoms such as anorexia, weight loss, and malaise. Acute
bacterial endocarditis typically occurs on native heart valves. It is most often caused by virulent
organisms such as S. aureus, beta-hemolytic streptococci, and less commonly by S. lugdunensis,
enterococci, and S. pneumoniae. Subacute bacterial endocarditis is usually caused by viridans
group streptococci, enterococci, and fastidious gram-negative rods from the oral cavity. Several
difficult-to-grow organisms are associated with “culture-negative” endocarditis. Prosthetic valve
endocarditis is often caused by coagulase-negative staphylococci but can also be caused by
S. aureus, other skin flora, enteric gram-negative rods, and fungi.
There are a number of risk factors for endocarditis, particularly in the acute form. These
include diabetes, alcoholism, intravenous drug use, malignancy, infections in other sites, and
immunosuppression. Anatomic defects also predispose patients to the development of infectious
endocarditis. Such defects include mitral valve prolapse, congenital or rheumatic heart disease,
and calcific aortic stenosis. The worst prognosis among patients with valvular disease is associated
with those who have aortic valve involvement. The mitral valve, however, is the most frequently
involved. Most individuals with endocarditis are between 45 and 60 years of age.
Diagnosis
The clinical and laboratory features of acute and subacute bacterial endocarditis are different
(Table 5–9). It should be noted, however, that patients can present with a syndrome of intermedi-
Laboratory confirmation ate severity between acute and subacute endocarditis, usually as a result of infection by organisms
of infective endocarditis of intermediate virulence, such as Enterococcus species, Haemophilus species, and the Streptococ-
usually involves isolation cus bovis group. Laboratory confirmation of infective endocarditis usually involves isolation of
of the same organism the same organism from multiple blood cultures. Organisms are more likely to be isolated from
from multiple blood blood cultures in patients who have acute bacterial endocarditis because they have a high-grade
cultures. Organisms are persistent bacteremia. If 3 sets of blood cultures are obtained, the blood cultures are positive in
more likely to be isolated more than 99% of patients who have not received antibiotics. At least 2 sets of blood cultures
from blood cultures in should be obtained by separate venipunctures at presentation. The erythrocyte sedimentation
patients who have acute
rate is a nonspecific test that is almost always elevated in cases of endocarditis, but it is useful
bacterial endocarditis
for monitoring the response to therapy. It is not uncommon to obtain negative blood cultures in
because they have a
patients who meet the clinical and echocardiographic criteria for infectious endocarditis. Many
high-grade persistent
bacteremia. of these “culture-negative” cases are due to prior antibiotic therapy. In the past, the “HACEK”
group of fastidious oral gram-negative rods was linked to culture-negative endocarditis, but these
organisms are readily detected by modern blood culture systems. More recently, interest has
focused on Coxiella burnetii, Bartonella spp., Tropheryma whipplei, and Brucella spp. as potential
causes of “culture-negative” endocarditis. These agents can be detected by a combination of sero-
logic and molecular methods.
TABLE 5–9 Evaluation of the Patient With Infective Endocarditis

Differentiating
Clinical Features Acute Infective Endocarditis Subacute Infective Endocarditis
Temperature >102°F <102°F
Osler nodes No Yes
Janeway lesions Yes No
Roth spots (in eye exam) No Yes
Laboratory tests
Organisms most often Highly virulent pathogens such as Staphylococcus Organisms of lower virulence such as viridans streptococci,
detected in blood culture aureus, Streptococcus pneumoniae, Pseudomonas enterococci, and Streptococcus bovis; many other organisms can
aeruginosa, often from a recognizable focus of cause infective endocarditis, but are difficult to identify because
infection they may require selective media or long periods for growth
Urinalysis and urine culture The presence of hematuria and a pathogenic Urinalysis reveals hematuria, pyuria, RBC casts, bacteriuria,
organism in a urine culture, in the appropriate and proteinuria
clinical context, is consistent with acute infective
endocarditic
Complete blood count findings
Erythrocyte ≥50 mm/h ≥50 mm/h

sedimentation rate
Anemia (normochromic, No Yes
normocytic)
Markedly elevated Yes No

WBC count
Transesophageal >90% sensitivity in detecting vegetations on >90% sensitivity in detecting vegetations on cardiac valves
echocardiography cardiac valves; it is also capable of identifying
valvular perforation, regurgitation, and abscess
formation in many patients
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM

Overview
Many organisms can produce an infection within the CNS. The major sites of infection are the Many organisms can
meninges and brain parenchyma. Most organisms gain access to the CNS by hematogenous produce an infection
spread or by direct extension from an adjacent site. Bacterial infections can cause acute men- within the CNS. The
ingitis or may lead to formation of a brain abscess. Viral infections can present as meningitis or major sites of infection
encephalitis, but often both sites are involved and the infection is more appropriately described are the meninges and
as meningoencephalitis. Both fungi and mycobacteria can cause chronic meningitis while sev- brain parenchyma.
eral parasites can cause intracerebral mass lesions. Each of these syndromes is often associated Most organisms gain
with specific organisms, information that can be used to guide the diagnostic workup. Rational access to the CNS by
hematogenous spread or
test ordering based on clinical presentation is particularly important in patients with CNS infec-
by direct extension from
tions since diagnostic specimens are difficult to obtain and are often present in limited quantities.
an adjacent site.
Table 5–10 provides information on organisms that are frequently described causes of meningitis
and/or encephalitis.
Acute Bacterial Meningitis

Description
Bacterial meningitis may present as a progressive illness over several days, or as a fulminant pro-
cess that develops within hours. There is no single clinical sign that is pathognomonic of meningi-
tis. Adolescents and adults typically present with combinations of fever, headache, nuchal rigidity,
and other meningeal signs, and a decreased level of consciousness that can range from lethargy to
coma; however, these findings are not present in all patients. Neonates and infants often present
TABLE 5–10 Laboratory Evaluation for Meningitis and Encephalitis

Age of Smear Used
Highest Higher Culture in Diagnosis Other Tests of
Disease/Organism Incidence Incidence in Available? of CSF Potential Use
Bacterial meningitis
Group B Streptococcus <1 month Neonates Yes Gram stain Rapid antigen detection,
(Streptococcus mainly useful for partially
agalactiae) treated infection
Streptococcus All ages >3 Hypogammaglobulinemia Yes Gram stain Rapid antigen detection,
pneumoniae months mainly useful for partially
treated infection
Escherichia coli and <1 month and Immunocompromised Yes Gram stain Rapid antigen detection for
other gram-negative >60 years E. coli K1, mainly useful for
bacteria (75% of partially treated infection
E. coli cases are
K1 strains)
Listeria monocytogenes <1 month and Immunocompromised Yes Gram stain

>60 years
Haemophilus influenzae 1 month to 5 Immunocompromised; Yes Gram stain Rapid antigen detection,
type b years unvaccinated mainly useful for partially
treated infection
Neisseria meningiditis 1 month Patients with complement Yes Gram stain Rapid antigen detection,
deficiencies mainly useful for partially
treated infection
Mycobacteria, especially ≥1 month Immunocompromised Yes Acid-fast stain Nucleic acid amplification
M. tuberculosis (rarely positive)
Treponema pallidum Adults Tertiary syphilis patients No None Several tests for syphilis
available (see the section
“Syphilis”)
Pseudomonas All ages Neurosurgical Yes Gram stain
aeruginosa postoperative patients
Staphylococcus aureus All ages Neurosurgical Yes Gram stain

postoperative patients
Coagulase-negative All ages Neurosurgical Yes Gram stain

staphylococci postoperative patients
Other streptococci All ages Neurosurgical Yes Gram stain

postoperative patients
Fungal meningitis
Cryptococcus Adults Immunocompromised Yes India ink Rapid antigen detection is

neoformans much more sensitive than
India ink
Coccidioides immitis Adults Immunocompromised; Yes Calcofluor Serologic testing of serum

those living in the smear and CSF
southwestern United
States, parts of Latin
America
Histoplasma capsulatum Adults Immunocompromised; Yes Calcofluor Serum, urine, or CSF antigen
those living in the smear test; serum test for antibody
Ohio and Mississippi to organism
River Valleys, and
parts of Central
America

TABLE 5–10 Laboratory Evaluation for Meningitis and Encephalitis (continued)

Age of Smear Used
Highest Higher Culture in Diagnosis Other Tests of
Disease/Organism Incidence Incidence in Available? of CSF Potential Use
Viral meningitis/encephalitis/meningoencephalitis
Enteroviruses (includes All ages Late summer and early fall Yes—viral None RT-PCR of CSF specimen is the
echovirus and including culture using preferred diagnostic test
coxsackievirus) infants throat swab,
and stool; CSF
culture less
sensitive than
RT-PCR
Herpes simplex virus-1 All ages Can be primary or Culture of None PCR of CSF specimen is
including reactivation infections CSF has the preferred diagnostic
infants low yield (can test; serologic testing;
be performed histochemical staining of
on brain brain biopsy
biopsy)
Herpes simplex virus-2 Neonates Infant of infected mother Culture of CSF None PCR of CSF specimen is
in neonates the preferred diagnostic
test; serum test for
antibody to virus
Cytomegalovirus All ages Immunocompromised Shell vial None PCR of CSF; antigen detection
culture of CSF in circulating WBCs; serum
or tissue test for antibody to virus
Arboviruses Peak age group Depending on Rarely useful None Serum and/or CSF tested for
varies for the virus in the group, antibody to specific viruses;
the different specific geographic RT-PCR available for some
viruses in this regions and seasons viruses (eg, West Nile)
group for higher infection
rates because
transmission is by
insects, usually mosquitoes
or ticks
Rabies virus All ages Individuals bitten or Rarely useful None Immunofluorescence of
scratched by rabies-prone brain biopsy specimen;
animal testing of serum and CSF for
antibodies to virus; RT-PCR
on saliva
Measles virus Childhood Nonvaccinated individuals Rarely useful None Testing of serum for antibodies
recently exposed to to virus
measles infection
Mumps virus Childhood Nonvaccinated individuals Yes—viral None Testing of serum for antibodies
recently exposed to culture of to virus
mumps infection throat swab,
CSF, and urine
HIV Adults Patients with AIDS or Rarely useful None Assays for diagnosis
unexplained opportunistic and monitoring of HIV
infections
Varicella zoster virus All ages Immunocompromised; Rarely useful None PCR of CSF specimen; testing
exposure to recent of serum for antibodies to virus
varicella zoster infection
Epstein–Barr virus Children, Recent exposure to Not available None PCR of CSF specimen; testing
adolescents, individual with infectious in clinical labs of serum for antibodies to virus
and young mononucleosis (research only)
adults
CSF, cerebrospinal fluid; PCR, polymerase chain reaction.
with nonspecific signs such as irritability, while nausea and vomiting are frequent complaints in
children. Confusion, often without fever, is a common presenting sign in the elderly.
In most cases, the bacteria responsible for meningitis are acquired through the upper respira-
tory tract and then invade the blood. From the blood, they can then seed the meninges. There are
a variety of factors that increase the risk for development of meningitis. These include splenec-
tomy, sickle cell disease, cerebrospinal fluid leak, fistula or shunt, recent neurosurgical procedure,
and infection contiguous to the CNS.
The organisms responsible for acute bacterial meningitis are highly dependent on the age of
the patient and the clinical setting. S. agalactiae (group B Streptococcus [GBS]), E. coli, or Listeria
monocytogenes are responsible for most cases of neonatal and infant meningitis. S. pneumoniae
and N. meningitidis are responsible for most cases of community-acquired bacterial meningitis in
children and adults (widespread vaccination for Haemophilus influenzae type b has nearly elimi-
nated this previous childhood scourge). Elderly patients are at increased risk of infection with
L. monocytogenes and aerobic gram-negative rods. In contrast, staphylococci and gram-negative
rods are major causes of CNS shunt infections and postneurosurgery nosocomial infections.
Knowledge of these patterns is important when deciding on empiric antibiotic therapy.
Diagnosis
Examination and culture of CSF is essential. There is usually a markedly elevated WBC count with
a preponderance of neutrophils, elevated protein, and decreased glucose (relative to the blood
level). Gram stain of CSF reveals the causative organism in 70% to 90% of cases of pneumococ-
cal and meningococcal meningitis. The percentage is generally lower for other bacteria. Bacterial
culture is essential in all cases because it has the greatest sensitivity and specificity. Patients who
have rapidly progressive or severe disease frequently receive a dose of antibiotics before a CSF
specimen can be collected. Although this may cause a false-negative culture, it should have little
or no effect on the cell count and differential, protein, glucose, and Gram stain. Immunoassays
that detect S. pneumoniae and N. meningitidis capsular antigens in CSF are useful in these patients
with partially treated meningitis, but they are not more sensitive than a Gram stain.
Acute Viral Meningitis

Description
Viral meningitis (often described as aseptic meningitis due to the absence of bacteria) presents
with fever, headache, and meningeal signs. There may be a mildly decreased level of conscious-
ness. At least 75% of these infections are caused by members of the enterovirus family that
includes the coxsackieviruses and echoviruses. Arboviruses (arthropod-borne viruses), HSV-2,
HIV, and many other viruses can also cause this syndrome. Both enterovirus and arbovirus infec-
Viral meningitis (often tions exhibit seasonal variation; most cases occur in the summer and early fall. Viral meningitis is
described as aseptic usually a self-limited illness with a generally good prognosis. This is fortunate since there are cur-
meningitis due to the rently no clinically useful antiviral drugs that are active against the enteroviruses and arboviruses.
absence of bacteria) The clinical diagnosis of viral meningitis is often not clear-cut because there can be parenchymal
presents with fever, involvement leading to varying degrees of encephalitis (see below). In addition, several other con-
headache, and meningeal ditions can cause a similar clinical syndrome. These include partially treated bacterial meningitis,
signs. At least 75% of neoplastic diseases that have spread to the meninges, and immune-mediated diseases. It is impor-
these infections are tant to identify these conditions because each of them is treatable and requires specific therapy.
caused by members Acute bacterial meningitis must be differentiated from viral and fungal meningitis. There is
of the enterovirus
a significant difference in the CSF findings between viral and bacterial meningitis (Table 5–11).
family that includes the
coxsackieviruses and
echoviruses. Diagnosis
CSF analysis usually reveals an elevated WBC count with a preponderance of mononuclear cells,
elevated protein, and a normal glucose. Identification of the causative agent is best achieved
through the use of specific nucleic acid amplification tests or immunoassays; routine viral culture
has a relatively poor yield in most cases. Many clinical or reference laboratories offer RT-PCR
assays for enteroviruses, HSV, and West Nile virus. Immunoassays for detection of IgM and IgG
are used to detect other viruses. Bacterial culture and cytopathology examination may be indi-
cated if the diagnosis is unclear.
TABLE 5–11 Typical Cerebrospinal Fluid (CSF) Findings in Meningitisa

Normal Bacterial Viral Fungal or Tuberculous
WBC (count/mL) 0-5 >100-5000 100-1000 50-500
Neutrophils (% of total WBC) 0-15 >80 <50 b

<50
Glucose (mg/dL) 45-65 <40 45-65 30-45
CSF/blood glucose ratio 0.6 <0.4 0.6 <0.4
Protein (mg/dL) 20-45 >150 50-100 100-500

a
Data from Segretti J, Harris AA. Acute bacterial meningitis. Infect Dis Clin North Am. 1996;10:797–809 and Derber CJ, Troy SB.
Head and neck emergencies: bacterial meningitis, encephalitis, brain abscess, upper airway obstruction, and jugular septic
thrombophlebitis. Med Clin North Am. 2012;96:1107–1126.
b
The percentage of neutrophils can be elevated during early stages of infection.
Chronic Meningitis
Description
Patients suffering from chronic meningitis usually present with a variety of signs including low-
grade fever, headache, lethargy, confusion, nausea, vomiting, and stiff neck that develop over a
period of 1 to 4 weeks. Fungi and mycobacteria are responsible for many cases of chronic meningi-
tis. The encapsulated yeast, C. neoformans, is 1 of the most common causes, particularly in patients
with depressed cell-mediated immunity due to HIV infection or immunosuppressive therapy.
C. neoformans is acquired by inhalation that usually causes an asymptomatic pulmonary infection.
These organisms then spread to the CNS by the hematogenous route. C. gattii, previously classi-
fied as a subspecies of C. neoformans, often causes meningitis in patients who are not infected with
HIV. Coccidioides immitis and C. posadasii, dimorphic fungi that are prevalent in the southwestern
United States, are also acquired by inhalation and have a predilection for infecting the meninges
and CNS. Immunosuppressed patients who harbor Mycobacterium tuberculosis are at increased
risk of CNS tuberculosis (TB). Neoplastic and immune-mediated diseases can also cause chronic
meningeal symptoms; it is important to distinguish between these conditions and infection.
Diagnosis
The diagnosis of chronic meningitis requires evaluation of the CSF. Typically there are an
increased number of mononuclear cells, mildly elevated protein, and normal glucose (except
in TB). Immunoassays for C. neoformans capsular polysaccharide can be performed in less than
an hour, have a very high sensitivity and specificity, and are superior to visual examination of
India ink preparations (the antigen test does not distinguish between C. neoformans and C. gattii).
Fungal culture should also be performed. If the patient is at increased risk of disseminated TB (ie,
purified protein derivative [PPD]-positive or a history of pulmonary TB), then the CSF should be
tested for mycobacteria. AFB smears are quite insensitive. PCR of CSF can provide early confir-
mation of M. tuberculosis infection in many cases. However, culture should still be performed to
obtain an isolate for susceptibility testing.
Encephalitis
Description
Viral encephalitis is an infection of the brain parenchyma that can produce permanent neuro-
logic damage or death in persons of all ages. A higher incidence of viral encephalitis is found in
young children, the elderly, and persons with impaired immunity. For some viruses, there is a
seasonal variation for infection. Most of the viruses that produce encephalitis enter the CNS via the
hematogenous route. In its mildest form, viral encephalitis can present with fever and headache,
and in its most severe form as an acute fulminating disorder with seizures and death. Prominent
clinical findings include altered level of consciousness, altered mental status, headache, seizures,
and other signs of neurologic dysfunction. The most important cause of sporadic encephalitis is
HSV-1 that often causes permanent neurologic damage or death. Fortunately, there is effective
Viral encephalitis is an antiviral therapy for HSV encephalitis that can prevent most of these complications if given early
infection of the brain in the infection. Arboviruses transmitted by mosquitoes are responsible for periodic epidemics of
parenchyma that can encephalitis. A dramatic example of this phenomenon was the appearance of West Nile virus in the
produce permanent eastern United States in the summer of 1999 and its subsequent spread across the country during
neurologic damage or the next 4 years. Arboviral encephalitis is usually a self-limited infection. Most patients recover, but
death in persons of all a significant number have persistent neurologic symptoms. Amebas such as Naegleria should also
ages. The most important be considered in the differential diagnosis of encephalitis since they require specific therapy.
cause of sporadic
encephalitis is HSV-1 that
often causes permanent
Diagnosis
neurologic damage or The diagnosis of viral encephalitis includes evaluation of CSF. In patients with viral encephalitis,
death. there is a predominantly lymphocytic pleocytosis, with a slight to moderate elevation of CSF
protein, and no change in glucose content from normal. However, there are variations, depend-
ing on the virus that produces the encephalitis. Some of the agents can produce CSF findings that
mimic those of bacterial meningitis (eg, pleocytosis with an increased number of neutrophils),
particularly during the early stages of infection. The diagnosis of HSV encephalitis should be
confirmed with a PCR assay of CSF that detects HSV DNA. This test is very sensitive and specific.
Because of the severity of HSV infections and the availability of effective treatment, it should be
performed on any patient with suspected encephalitis. Viral culture of CSF is insensitive and
should not be routinely performed. Encephalitis caused by other viruses is often diagnosed by
detection of viral nucleic acid or virus-specific IgM and IgG in serum or CSF.
Table 5–10 presents the laboratory evaluation for meningitis and encephalitis by disease
and/or organism. Organisms not listed in the table also may cause CNS infections. Chapter 2
provides descriptions of many of the tests mentioned in the table.
Brain Abscess
Description
A brain abscess is a focal lesion and therefore presents differently from meningitis and encephali-
tis. The most common clinical presentation is persistent, worsening headache. More than half of
patients will have focal neurologic deficits, but only half have fever. Bacterial abscesses can result
from hematogenous dissemination or extension from an adjacent site. The most common organisms
are viridans group streptococci, Haemophilus spp., and anaerobic gram-negative rods. If the patient
is immunosuppressed, then the abscess could be caused by Aspergillus and other fungi, Nocardia spp.
and Mycobacterium spp., and Toxoplasma gondii. Neurocysticercosis, a mass lesion that results from
infection with the pork tapeworm Taenia solium, often presents as new-onset seizures in an adult.
Diagnosis
Unlike other CNS infections, examination of CSF is unlikely to yield useful information and even
performing a lumbar puncture may be contraindicated. The initial diagnosis is usually based on
CT and MRI findings. Identification of the causative agent is important for guiding therapy. This
usually requires stereotactic biopsy of the abscess to obtain material for microscopic examination
and culture. Serologic assays performed on serum may be helpful in the diagnosis of Toxoplasma
infections and neurocysticercosis.
BONE INFECTIONS/OSTEOMYELITIS
Description
Osteomyelitis is an infection of the bone characterized by progressive, inflammatory destruction
of the bone tissue. It can be classified by the route of infection (hematogenous, contiguous spread,
direct traumatic, or surgical inoculation), the site of infection, the type of patient, or duration of
infection (acute or chronic). Hematogenous osteomyelitis is most common in prepubertal chil-
dren, where it usually involves the long bones, and older adults where it usually involves the ver-
tebrae. Less common sites of osteomyelitis include the sternoclavicular and sacroiliac joints, and
symphysis pubis. Children with acute osteomyelitis appear ill with fever, chills, localized pain, and
leukocytosis. In contrast, adults with vertebral osteomyelitis often have a subacute course with
TABLE 5–12 Organisms Associated With Osteomyelitis and Populations at Risk

Organism Population With Highest Incidence or Predisposing Condition
Staphylococcus aureus All ages, including infants and children; most frequent organism causing
hematogenous osteomyelitis
Salmonella spp. Sickle cell disease patients; immunocompromised individuals
Pseudomonas aeruginosa Intravenous drug abusers; those with a puncture wound to the foot;
patients with urinary catheters
Aerobic gram-negative rods Urinary tract infections, diabetic foot infections, or vascular insufficiency
(eg, Enterobacter and Proteus)
Aerobic streptococci Patients with bites, diabetic foot lesions, or vascular insufficiency
Anaerobic streptococci Patients with foreign body-associated infections such as those induced
by prosthetic joints (chronic infection), bites, diabetic foot lesions, or
decubitus ulcers
Mycobacterium tuberculosis Patients with a history of pulmonary tuberculosis; immunocompromised

individuals
Fungal species (includes Candida Patients with catheter-related fungemia; intravenous drug abusers;
and Aspergillus) immunocompromised individuals
slowly worsening back pain and little or no fever or leukocytosis. Hematogenous osteomyelitis is Osteomyelitis caused
usually caused by a single organism. S. aureus accounts for half of cases. Other frequently encoun- by a contiguous focus of
tered organisms are streptococci and enterobacteriaceae in neonates, gram-negative rods in the infection often results
elderly, Salmonella spp. in patients with sickle cell disease, P. aeruginosa in intravenous drug users, from an injury associated
and Candida spp. in patients with intravascular catheters. TB and brucellosis can cause vertebral with an open fracture
osteomyelitis in patients who have been exposed to these organisms. or following surgery for
Osteomyelitis caused by a contiguous focus of infection often results from an injury associ- reconstruction of bone.
ated with an open fracture or following surgery for reconstruction of bone. It is also common
in patients with poorly controlled diabetes mellitus due to peripheral neuropathy and vascular
insufficiency. This form of osteomyelitis is found almost exclusively in the foot and starts insidi-
ously in areas of traumatized skin. The infection of the skin may be easily overlooked as the
organism makes its way to the bones in the toes, metatarsal heads, and tarsal bones. Unlike hema-
togenous osteomyelitis, these contiguous focus infections are usually polymicrobial involving
combinations of staphylococci, streptococci, enterococci, and gram-negative rods. Additional
classes of organisms may be present in the wound if the injury site was contaminated with soil.
With between 500,000 and 1,000,000 hip replacements per year worldwide, infections associated
with prosthetic joints are also common (see the section “Infections of the Joints”).
Diagnosis
Imaging techniques can be used to detect osteomyelitis in the early phase and reveal the extent of
damage to bones and joints. Definitive diagnosis requires biopsy and culture of the infected tissue
in most cases. This permits an accurate identification of the organisms responsible for the osteo-
myelitis. In some situations, the organisms suspected of causing osteomyelitis may be identified
from the culture of synovial fluid, blood, or biopsy of contiguous lesions. Microbiologic culture
of blood or contiguous tissue does not definitively identify the organism in the bone, but it may
provide a strong indication of the organisms responsible for the osteomyelitis.
Table 5–12 lists the organisms most likely to cause osteomyelitis and identifies the popula-
tions at risk for infection by the named organisms.
INFECTIONS OF THE JOINTS

Description
Acute pain and swelling in the joint can be produced by infectious agents, crystals of monoso-
dium urate or other compounds, and a variety of less common causes. Because some organisms
The mainstays of the can destroy cartilage in a matter of days, the diagnosis of infectious arthritis must be made
laboratory investigation quickly. Organisms can seed the joint through the hematogenous route (via intravenous drug
for a joint infection are abuse, indwelling catheters, endocarditis), through direct inoculation from intra-articular injec-
synovial fluid Gram stain tions or arthroscopy, or from a contiguous site of infection, especially the bones and bursae.
and culture to identify Septic arthritis can resemble a variety of noninfectious processes, but an acutely inflamed joint
infecting organisms, and should be considered septic until proved otherwise. Septic arthritis is most often monoarticular
polarized microscopy with polyarticular involvement in less than 20% of the cases.
of the synovial fluid to
identify crystals in crystal-
induced arthritis. Diagnosis
The mainstays of the laboratory investigation for a joint infection are synovial fluid Gram stain
and culture to identify infecting organisms, and polarized microscopy of the synovial fluid to
identify crystals in crystal-induced arthritis. It should be noted, however, that crystal-induced
and infectious arthritis can coexist. Synovial fluid WBC counts consistent with infection are
≥50,000/μL, with more than 90% as neutrophils. Table 5–13 summarizes the organisms associ-
ated with joint infection and the relevant clinical features for each infection.
TABLE 5–13 Evaluation for Organisms Associated With Infections of the Joints
Population With Highest Incidence
Organism and/or Predisposing Conditions Clinical Features Laboratory Tests
Staphylococcus aureus Damaged joint; cutaneous abscess; Most common cause of septic Gram stain of synovial fluid is positive
intravenous drug abuse; prosthetic arthritis; can produce rapid in majority of cases
joint destruction of the joint
Selected streptococcal Diabetes mellitus Second most common cause of Gram stain and culture of synovial fluid
species (excluding septic joint after S. aureus; from and blood cultures reveal infecting
Streptococcus pneumoniae) benign to severe, depending organism in majority of cases
on the specific organism and
predisposing conditions
Coagulase-negative Prosthetic joint Inflammation and tenderness Gram stain and culture of synovial
staphylococci around a prosthetic joint fluid, with blood cultures, may reveal
organism
Neisseria gonorrhoeae Young adults May have genitourinary findings Gram stain of synovial fluid is positive in
of gonococcal infection; 25%-30% of cases; synovial fluid culture
dermatitis and synovitis is positive in 25%-50% of cases; blood
not uncommon culture is positive in 10%-15% of cases
Gram-negative bacilli Immunocompromised patients; Up to 20% of septic arthritis cases Gram stain of synovial fluid reveals
(pathogens include urinary or biliary tract infection; are caused by gram-negative organisms in about 50% of cases; culture
Pseudomonas aeruginosa, intravenous drug abusers (especially organisms of synovial fluid and blood also may lead
Serratia, Klebsiella, and for Pseudomonas); prosthetic joint; to organism identification
Enterobacter, which may be SLE and sickle cell disease (especially
specific for certain joints) for Salmonella)
S. pneumoniae Splenic dysfunction Accounts for <5% of septic Gram stain and culture of synovial fluid
arthritis cases and blood cultures reveal infecting
organism in majority of cases
Mycobacterial species Earlier tuberculous infection Up to 50% of patients with Culture of synovial fluid or synovial
(includes M. tuberculosis reactivated by age or M. tuberculosis joint involvement tissue may reveal organism
and M. marinum) immunosuppression also have pulmonary tuberculosis
Fungal species (pathogens Alcoholism; myeloproliferative Sporothrix is the most common Repeated cultures of synovial fluid
include Sporothrix, disorders cause of fungal arthritis; and tissue are often required for
Cryptococcus, Blastomyces, Cryptococcus and Blastomyces identification of the organism; a
Coccidioides, and Candida) infections may be associated calcofluor fungal smear of synovial
with adjacent osteomyelitis; fluid may be helpful
blastomycosis arthritis may
be associated with pulmonary
infection
Borrelia burgdorferi (Lyme Patients with Lyme disease or history Intermittent attacks of swelling One of several tests for Lyme disease
disease agent) of tick exposure in a large joint (see the section “Lyme Disease”)
SLE, systemic lupus erythematosus.
INFECTIONS OF THE SKIN AND ADJACENT SOFT TISSUE

Overview
Many different organisms can produce infections of the skin and soft tissue. Clinical manifesta-
tions and severity vary widely and include aggressive, fast-moving infections such as necrotizing
fasciitis, abscesses that require incision and drainage, chronic superficial fungal infections, and
rashes that can be caused by local or systemic viral infections. A brief description of infections
associated with particular organisms is provided in Table 5–14. The diagnostic information to
identify infecting organisms is also included in the table. Lyme disease and Bartonella infections
are discussed in Tables 5–15 and 5–16. A number of other infectious diseases, such as syphilis
Abscesses, infected
and herpes simplex infections, also have skin manifestations and are more fully described else-
wounds, and cellulitis are
where in this chapter. common acute infections
of the skin. The most
Acute Bacterial Infections common organisms are
S. aureus, beta-hemolytic
Abscesses, infected wounds, and cellulitis are common acute infections of the skin. The most streptococci (groups A
common organisms are S. aureus, beta-hemolytic streptococci (groups A and B), other strepto- and B), other streptococci
cocci and staphylococci, and several gram-negative rods. Certain underlying conditions, such as and staphylococci, and
diabetes and peripheral vascular disease, predispose to polymicrobial infections involving gram- several gram-negative
positive cocci and gram-negative rods. rods.
TABLE 5–14 Selected Skin and Soft Tissue Infections Caused by Bacteria
Skin/Soft
Tissue Predisposing
Infection Description Condition(s) Etiologic Agent(s) Clinical Findings Laboratory Diagnosis
Superficial Infection of hair Poor hygiene, Staphylococcus aureus Single or multiple Diagnosis usually made
folliculitis follicles of the skin occupational exposure most common superficial, dome- clinically; Gram stain
to oils and solvents shaped, pruritic and bacterial cultures
pustules at the ostium support the clinical
of hair follicles on the diagnosis
scalp, back, and/or
extremities
Hot tub Infection of hair Whirlpools and hot Pseudomonas Small erythematous Clinical diagnosis
folliculitis follicles of the skin tubs with low chlorine, aeruginosa pruritic papules topped supported by bacterial
high pH, and high by pustules in areas culture and Gram stain
water temperatures submerged in hot water of infected pustule or
water source
Furuncle Acute bacterial Skin areas subject S. aureus Indurated, dull red, Diagnosis usually
infection of to friction and tender nodule with made clinically; Gram
perifollicular perspiration, poor central purulent core stain and culture of
skin, usually hygiene, occupational on the face, buttocks, suppurative lesion
from preexisting exposure to grease perineum, breast, support the clinical
folliculitis or oil, malnutrition, and/or axilla diagnosis
alcoholism, and
immunosuppression
Carbuncle Coalescence of For untreated S. aureus Multiple abscess Diagnosis usually

interconnected furuncles, formations separating made clinically; Gram
furuncles; involves complications connective tissue stain and culture of
subcutaneous tissue include bacteremia, septae with drainage suppurative lesion
with drainage at endocarditis, and to surface along hair support the clinical
multiple sites osteomyelitis follicles diagnosis
Paronychia Infection of the Minor trauma causing Acute: staphylococci, Tender, red, swollen Clinical diagnosis
nail folds break in the skin, as beta-hemolytic areas extending around supported by bacterial
produced by splinters streptococci, gram- the nail fold with or and/or fungal cultures
Chronic: frequent negative enteric without pus of infected areas
immersion of hands bacteria
in water Chronic: Candida
albicans

TABLE 5–14 Selected Skin and Soft Tissue Infections Caused by Bacteria (continued)
Skin/Soft
Tissue Predisposing
Impetigo Localized purulent Children (2-5 years old) Group A beta-hemolytic Small superficial Clinical diagnosis
contagiosa infection of the skin living in warm, humid streptococci and vesicles that form supported by culture/
(nonbullous) climates; poor hygiene; S. aureus pustules rupture, Gram stain of base of
preexisting superficial forming characteristic early lesion positive
abrasions from insect yellow-brown, “honey- for staphylococci
bites, trauma, and colored” crusted lesions and/or streptococci;
other causes anti-DNase B and
antihyaluronidase titers
may be elevated
Bullous Localized purulent Occurs in newborns Usually due to S. aureus Begins as small vesicles Clinical diagnosis
impetigo infection of the skin and younger children producing exfoliative that quickly enlarge to supported by culture/
causing bullous on nontraumatized toxins form bullae with clear Gram stain of base of
lesions skin of the buttocks, fluid that rupture and lesion or clear fluid
perineum, trunk, and/ leave a brownish black from bullae showing
or face crust staphylococci
Staphylococcal Widespread bullae Higher incidence S. aureus producing Scarlatiniform rash with Diagnosis usually made
scalded skin and exfoliation in newborns and exfoliative exotoxins widespread tender clinically
syndrome as a severe younger children bullae with clear fluid;
(SSSS) manifestation bullae rupture, resulting
of an infection in separation of skin;
by S. aureus exfoliation exposes
strains producing large areas of red skin
exfoliative exotoxins
Ecthyma A variant of May occur de novo Usually group A beta- “Punched-out” ulcers Clinical diagnosis
impetigo on the or secondary to hemolytic streptococci with yellow crust supported by culture
lower extremities preexisting superficial extending into dermis, and Gram stain of base
causing punched- abrasions such as insect typically on lower of lesion that is positive
out ulcerative bites; occurs in children extremities for streptococci
lesions and elderly most often
Erythrasma Superficial chronic More common in Corynebacterium Slowly spreading Gram-stained imprints
bacterial infection males, obese patients, minutissimum pruritic, red brown of skin lesions reveal
of the skin and patients with macules with scales— gram-positive bacilli;
diabetes mellitus affecting axillae, groin, examination of skin
and toes under Wood’s lamp
reveals distinctive red
coral fluorescence
Erysipelas Acute inflammation Occurs most often Group A beta- 5%-20% facial, 70%- Difficult to culture
of superficial in infants, young hemolytic streptococci; 80% lower extremity; group A streptococci
layers of the skin children, and elderly; rarely, group B, C, painful, bright red, from lesion; up to
with lymphatic those with skin ulcers, G streptococci and edematous, indurated 20% of throat cultures
involvement local trauma/abrasion, S. aureus lesions with raised positive for group A
and eczematous border, well demarcated streptococci; blood
lesions; increased from uninvolved culture positive in 5%
susceptibility in skin; regional of cases
sites with impaired lymphadenopathy
lymphatic drainage common
Cellulitis Diffuse suppurative Occurs in sites of Nonimmunosuppressed Localized, painful, Gram stain/culture of
inflammation of skin previous tissue hosts: commonly erythematous, purulent exudate from
and subcutaneous damage such as group A beta- warm lesions, advancing edge may
tissues operative wounds, hemolytic streptococci; poorly demarcated reveal etiologic agent;
ulcers, and focal less commonly from uninvolved blood cultures positive
trauma; increased S. aureus; group B skin; regional in 25% of cases
incidence in and G streptococci in lymphadenopathy may
intravenous drug patients with lower be present; bacteremia
abusers extremity edema; and gangrene may
gram-negative rods in occur if untreated
immunosuppressed
patients; Pasteurella in
cat and dog bites

Skin/Soft
Tissue Predisposing
Synergistic A variant of Diabetes mellitus, Mixture of anaerobes Acute onset of tender Culture/Gram stain of
necrotizing necrotizing fasciitis obesity, advancing (anaerobic streptococci skin ulcers in lower exudate aspirated from
cellulitis (see following age, cardiac, and renal and Bacteroides most extremity draining foul- lesion
(nonclostridial entities) involving disease commonly) and smelling, red-brown
anaerobic skin, muscle, facultative bacteria (“dishwater”) pus, with
cellulitis) subcutaneous (Klebsiella, E. coli, underlying gangrene
tissue, and fascia Proteus) of subcutaneous tissue
and muscle; tissue gas
in 25% of patients;
systemic toxicity is
significant
Necrotizing A deep-seated, Diabetes mellitus, At least 1 anaerobic Sudden onset of tender, Surgical exploration
fasciitis (Type I) severe necrotizing alcoholism, parenteral species (most warm, erythematous, required to distinguish
infection of the drug abuse; occurs at commonly Bacteroides well-demarcated from cellulitis;
subcutaneous sites of trauma such or Peptostreptococcus) cellulitis, usually leukocytosis,
tissue, resulting as an insect bite, and along with a facultative involving the lower thrombocytopenia,
in progressive following laparotomy anaerobic species extremity, abdominal azotemia, and increased
destruction of performed in the such as streptococci or wall, perianal, and/or serum levels of creatine
superficial and, in presence of perineal gram-negative enteric groin areas; sequential kinase (CK) may be
some cases, deep soiling, decubitus bacilli such as E. coli, skin color changes present; Gram-stained
fascia and fat ulcers, and perirectal Enterobacter, Klebsiella, from red-purple to smears of exudates
abscesses and Proteus patchy blue-gray over reveal a mixture of
several days; within 3-5 organisms; blood
days, skin breakdown cultures are frequently
occurs with bullae, a positive; subcutaneous
seropurulent exudate, gas and soft tissue
frank cutaneous swelling detectable on
gangrene, and skin radiographs
anesthesia; high fevers
and systemic toxicity
with early shock and
organ failure common
Necrotizing A deep-seated, Occurs in 50% Group A streptococci, Sudden onset of tender, Surgical exploration
fasciitis (Type severe, necrotizing of patients with either alone or in warm, erythematous, required to distinguish
II) (also known infection of the streptococcal toxic combination with well-demarcated from cellulitis;
as hemolytic subcutaneous shock syndrome; other species, most cellulitis, usually leukocytosis,
streptococcal tissue, resulting predisposing factors commonly S. aureus involving the lower thrombocytopenia,
gangrene) in progressive also include diabetes extremity, abdominal azotemia, and
destruction of mellitus, long-term wall, perianal, and groin increased serum levels
superficial and, in steroid therapy, areas; sequential skin of CK may be present;
some cases, deep cirrhosis, peripheral color changes from Gram-stained smears of
fascia and fat vascular disease, red-purple to patchy exudate reveal gram-
a recent history of blue-gray over several positive cocci in chains;
minor trauma, stab days; within 3-5 days, surgical debridement
wounds, and surgical bullae develop with provides tissue for
procedures seropurulent exudate, culture and Gram stain;
frank cutaneous subcutaneous gas and
gangrene, and skin soft tissue swelling
anesthesia; high fevers present on radiograph
and systemic toxicity
with early shock and
organ failure
Clostridial A necrotizing Dirty or inadequately Clostridial species, Localized edema of Gram stain of drainage
anaerobic clostridial infection debrided traumatic usually Clostridium wound site; thin, foul- shows numerous blunt-
cellulitis of devitalized wounds; preexisting perfringens smelling drainage of ended, thick, gram-
subcutaneous localized infection; wound with minimal positive bacilli and
tissue with rare contamination of pain, extensive gas variable numbers of
involvement of surgical wounds formation in tissues, neutrophils; soft tissue
deep fascia or and frank crepitant radiographic films show
muscle cellulites abundant gas

Skin/Soft
Tissue Predisposing
Clostridial Rapidly progressive Wounds associated C. perfringens accounts Sudden onset of Surgical exploration
myonecrosis infection with trauma and for 80% of cases; severe pain at the critical in
(gas gangrene) characterized by open fractures such other species include site of a wound with demonstrating
muscle necrosis and as gunshot wounds; C. septicum, C. novyi, rapid progression to devitalized muscle
systemic toxicity intestinal and biliary and C. sordelli; the localized tense edema tissue; CT scan shows
caused by potent tract surgery toxins released by and pallor; crepitance gas in the muscle and in
clostridial exotoxins these organisms are is a late finding and is fascial planes with soft
responsible for much neither a sensitive nor tissue swelling; Gram
of the morbidity and a specific feature; as stain of exudate shows
mortality associated the lesion progresses, typical gram-positive
with these infections the skin progresses or gram-variable rods
to magenta or brown with spores and lysed
discoloration with or absent neutrophils;
brown serosanguinous with C. perfringens,
discharge and “mousey” organism shows typical
odor boxcar appearance
without spores on
Gram stain, “double-
zone hemolysis” on
anaerobic blood
plate, and lecithinase
activity (alpha-toxin);
elevated CK, LDH, and
myoglobin due to
myonecrosis
Spontaneous Rapidly progressive Hematologic Most cases due to Sudden onset of pain Surgical exploration
or infection malignancies, colon C. septicum and localized swelling critical in
nontraumatic characterized by cancer, diabetes of extremity, followed demonstrating
gas gangrene muscle necrosis and mellitus, peripheral by discoloration, myonecrosis; CT scan
systemic toxicity vascular disease; blister formation, and shows gas in the
caused by clostridial commonly with no crepitance; associated muscle and fascial
infection obvious portal of fever, abdominal pain, planes with soft tissue
entry; not associated vomiting, and diarrhea swelling; Gram stain
with traumatic or of exudate shows
surgical wounds typical gram-positive
or gram-variable rods
with spores and lysed
or absent neutrophils;
elevated CK, LDH, and
myoglobin due to
myonecrosis
CT, computed tomography; LDH, lactate dehydrogenase.
Cat/dog and human bite wounds are commonly infected with the above organisms, but they
are also associated with specific organisms (Pasteurella multocida and Eikenella corrodens, respec-
tively).
Necrotizing fasciitis (usually caused by S. pyogenes) and clostridial myonecrosis (also known
as gas gangrene) are uncommon, but they are life-threatening infections that require prompt sur-
gical and medical intervention. Traumatic gas gangrene is usually caused by contamination of
wounds by Clostridium perfringens (an anaerobic spore-forming gram-positive rod). Spontane-
ous gas gangrene is usually caused by C. septicum, often in patients with underlying gastrointesti-
nal malignancies or neutropenia.
Lyme Disease
Description
The causative agent of Lyme disease is Borrelia burgdorferi, which is spread by the bite of a tick of
the genus Ixodes. Lyme disease is the most common vector-borne infection in North America and
TABLE 5–15 Laboratory Evaluation for Lyme Disease

Enzyme-linked immunoassay Detects serum IgM and IgG that react with a sonicated extract of B. burgdorferi.
(ELISA)—total antibodies Used as a screening test; because of the potential for false-positive reactions
a positive EIA is followed by Western blot in the CDC-recommended 2-step
algorithm. EIA can also be used to determine CSF/serum indices
Indirect immunofluorescence This assay also detects serum antibodies against B. burgdorferi. It has been
assay (IFA) replaced by EIAs in most laboratories
Western blot analysis This assay detects serum antibodies to specific antigens of B. burgdorferi as a
qualitative yes/no answer. The results should be interpreted according to CDC
guidelines: a positive IgG blot is defined as the presence of antibodies that
react with at least 5 out of 10 specific proteins; a positive IgM blot is defined as
the presence of antibodies that react with at least 2 out of 3 specific proteins.
Western blots can also be performed on CSF
Anti-C6 ELISA A recently developed ELISA that detects antibodies against a highly
immunogenic, highly conserved epitope of B. burgdorferi. More sensitive than
the 2-step algorithm (because of low sensitivity of the Western blot in early
Lyme disease) but slightly less specific than the 2-step algorithm
PCR analysis May be useful for testing CSF or joint fluid in selected cases
in Europe. It tends to go unnoticed by many patients because the associated influenza-like symp- The causative agent of
toms are not specific for the disease. Within days to weeks of infection, a distinctive skin rash, Lyme disease is Borrelia
known as erythema migrans, appears. It is important to treat the patient at the time that this rash burgdorferi, which is
appears to prevent subsequent potential neurologic, cardiac, or musculoskeletal complications. spread by the bite of a
tick of the genus Ixodes.
Lyme disease is the
Diagnosis
most common vector-
Unlike many infections, this laboratory diagnosis is rarely based on culture of B. burgdorferi. This borne infection in North
is because there are few organisms in clinical specimens, and these organisms require specialized America and in Europe.
media for growth with prolonged incubation. Therefore, the diagnosis rests upon a characteristic
clinical picture supported by positive serologic tests consistent with the infection. In patients who
present with erythema migrans, the diagnosis is straightforward. However, for many patients with
less specific symptoms and equivocal serologic test results, it is difficult to make a definitive diag-
nosis. Serologic testing for Lyme disease is a 2-step process involving a screening enzyme-linked
immunoassay (ELISA), followed by a confirmatory immunoblot (see Chapter 2 for a description
TABLE 5–16 Evaluation for Cat-scratch Disease and Bacillary Angiomatosis

Predisposing
Infection Description/Clinical Findings Conditions Etiologic Agents Laboratory Diagnosis
Cat-scratch disease Regional lymphadenopathy that History of contact Most cases Lymph node biopsy with
develops 2-3 weeks after contact with with cat are caused by characteristic appearance (stellate
a cat in the presence of a scratch or eye Bartonella henselae necrotizing granulomas); Warthin–
lesion; usually persists for 2-4 months (rare causes include Starry silver stain may reveal bacilli,
other Bartonella but only in a small percentage of
spp. and Afipia felis) specimens; diagnosis is supported by
detection of antibodies to B. henselae
in serum; PCR for B. henselae DNA
useful in atypical cases
Bacillary A disease with proliferative vascular AIDS patients, B. henselae Skin biopsy with a characteristic
angiomatosis lesions caused by infection with small especially those (from cats) and vascular proliferation and numerous
gram-negative organisms of the genus with low CD4 cell B. quintana bacilli detected on Warthin–Starry
Bartonella; cutaneous lesions: papular counts; history of (transmitted by stain; organisms can be isolated from
red nodules; bacillary angiomatosis contact with cat lice) blood using special isolator tubes
lesions also can occur in bones, liver, or exposure to lice and grown in culture
spleen, CNS, and other sites
CNS, central nervous system; PCR, polymerase chain reaction.
of these assays). There are several important issues regarding the serologic assays to detect Lyme
disease:
• The serologic tests are not entirely specific for Lyme disease.
• Serum samples collected in the early stage of the disease may contain no antibodies to
the organism because there is little or no antibody production in many patients until 3 to
4 weeks after the onset of the illness.
• The immune response is variable, and treatment with antibiotics can reduce the magnitude
of the response.
Cross-reactivity between the antigens from B. burgdorferi and antigens from other organisms
may occur. For example, false-positive serologic reactions for Lyme disease have been reported
in patients with RMSF, leptospirosis, and syphilis. False-positive reactions can also occur in some
patients with autoimmune diseases.
PCR-based assays for detection of B. burgdorferi DNA may be useful for testing joint fluid
or CSF. Only properly validated assays should be used since in a low-prevalence population (ie,
patients with nonspecific symptoms and no history of a tick bite) the positive predictive value can
be very low. A summary of the different assays currently available for diagnosis of Lyme disease
is provided in Table 5–15.
Cat-scratch Disease and Bacillary Angiomatosis

Descriptions
Several organisms of the genus Bartonella were found to be associated with human disease in
the 1990s when they were identified as the etiologic agents in cat-scratch disease and bacillary
angiomatosis. Most cases of cat-scratch disease are caused by B. henselae (other Bartonella spp.
and Afipia felis may account for a small percentage of these cases). Initially a papule or pus-
tule forms at the site of the scratch, but most individuals seek medical attention several weeks
Several organisms of the later because of the development of a regional lymphadenopathy (mainly in the neck or upper
genus Bartonella were extremity). In most cases, it resolves spontaneously, although in rare cases severe complications
found to be the etiologic can occur, including encephalitis, conjunctivitis, and neuroretinitis. Bacillary angiomatosis is a
agents in cat-scratch disorder in which there are distinctive and potentially lethal vascular proliferative responses in
disease and bacillary the skin, the bones, and other organs. Bacillary angiomatosis is most commonly diagnosed in
angiomatosis. individuals infected with HIV.
Diagnosis
A further description of cat-scratch disease and bacillary angiomatosis and recommendations for
their diagnosis are included in Table 5–16. Serologic tests and PCR-based tests may be useful to
support the histopathologic findings from lymph node biopsies in cat-scratch disease and from
skin biopsies in bacillary angiomatosis. Bartonella spp. are difficult to culture.
Fungal Infections
Fungal infections of the skin can be characterized as superficial, cutaneous, and subcutaneous.
Infections caused by fungi such as Malassezia spp. are limited to the superficial layers of the skin
and can result in patchy alteration of pigmentation. The more invasive cutaneous infections
caused by dermatophytes and subcutaneous infections are discussed in the following sections.
Descriptions
The dermatomycoses (also known as ringworm) are skin infections caused by dermatophytes.
These organisms are closely related fungi that invade keratinous tissues such as skin, hair,
and the fur of animals. The dermatophytes are classified into 3 genera, Epidermophyton,
Microsporum, and Trichophyton. Dermatophyte infections are named according to the ana-
tomic location (in Latin) for the body site, following the word “tinea.” For example, tinea
pedis is a dermatophyte infection of the foot. The diagnosis of a dermatophyte infection is
made by microscopic examination of a scraping of the lesion and by culturing the specimen
on selective agar that inhibits the growth of commensal bacteria and other fungi. When organ-
isms are successfully grown in culture, they are identified to the species level by colony and
microscopic morphology.
Sporotrichosis is a chronic infection characterized by nodular lesions in cutaneous and
subcutaneous tissues and adjacent lymphatics. This infection is caused by Sporothrix schenckii,
a dimorphic fungus that is typically introduced by traumatic implantation into the skin (eg, dur-
ing gardening). Sporotrichosis commonly displays a lymphocutaneous pattern as it tracks up the
lymphatic system in the hand and arm. Rare manifestations include pulmonary and disseminated
infections. Histologic examination of a specimen will often reveal granulomatous inflammation,
but organisms are rarely seen. Therefore, diagnosis usually depends on culturing the specimen to
permit microbiologic isolation of the S. schenckii organisms.
Mycetoma is a chronic infectious disease that involves cutaneous and subcutaneous tissues,
fascia, and bone, and remains localized. It is characterized by draining sinuses, with aggregates of
the etiologic agent in the pus draining from the sinuses. The fungi that produce the mycetomata
are associated with woody plants and soil. The organisms are usually introduced by traumatic
inoculation into the skin. A tumor-like deforming disease can develop during subsequent years
if untreated. Dozens of fungal organisms have been documented as causes of mycetoma. In the
United States, the most common agent is Pseudoallescheria boydii. The asexual form of this organ-
ism is known as Scedosporium apiospermum. Other fungi predominate in tropical and subtropical
areas. The fungal elements are most often found in the center of a suppurative and granulomatous
lesion that develops in a deep site and extends out to the skin for drainage. Draining sinuses
appear in essentially all patients within 1 year of the initial trauma. Identification of the causative
agent is made by fungal smear and culture of the material draining from the sinus tracts. Clini-
cally similar infections are caused by filamentous gram-positive bacteria in the genus Nocardia.
It is important to identify the infecting agent because the therapy for fungi is very different from
that used for bacteria.
Chromomycosis is a subcutaneous infection by organisms originating in the soil, with only
rare cases of dissemination. It is most often seen in tropical or subtropical environments and is
rare in the United States. The lesions typically appear on the lower extremities. They are pink,
scaly papules that expand to form a superficial nodule, and their presence suggests the diagnosis.
Examination of the lesions microscopically in potassium hydroxide (KOH) or calcofluor white
preparations can be diagnostic. Without therapy, which is usually surgical, the scaly papules grow
to form nodules with a verrucous and friable surface.
Diagnosis Primary infection with

A further description of the infections and the tests used to identify the associated organisms are varicella zoster virus
included in Table 5–17. causes chickenpox, a
vesicular rash that occurs
predominantly on the
Viral Infections With Prominent Skin Manifestations trunk, scalp, and face.
This disease is usually
Overview self-limited, with the
Viral infections can cause a variety of macular, papular, or vesicular rashes. Many of these typi- symptoms resolving after
cally occur in the pediatric age range and may be associated with systemic signs and symptoms. 7 days.
enteroviruses and parvovirus B19 are discussed in other sections.
Varicella Zoster Viral Infection

Description
Primary infection with VZV causes chickenpox, a vesicular rash that occurs predominantly on
the trunk, scalp, and face. This disease is usually self-limited, with the symptoms resolving after
7 days. Varicella can also produce pneumonia, with pulmonary symptoms manifested approxi-
mately 4 days after the varicella rash. After primary infection, the virus enters the latent phase
and remains in sensory ganglia. On reactivation, which occurs in a minority of adults, it produces
herpes zoster, a vesicular rash that occurs along a dermatome distribution. The incidence of vari-
cella has been declining since the introduction of a live virus vaccine.
TABLE 5–17 Evaluation for Superficial, Cutaneous, and Subcutaneous Fungal and Mycobacterial Infections
Disease/
Dermatomycosis Etiologic Agent(s) Clinical Findings Anatomic Pathology Microbiology
Tinea versicolor Malassezia furfur Hypopigmented or Skin biopsies may Round yeast forms and short
(pityriasis) hyperpigmented macules on demonstrate yeast hyphae visible by direct
trunk or proximal limbs forms with short hyphae microscopy of lesion scrapings
Tinea capitis (scalp ring Trichophyton, Pruritic, scaly, erythematous Skin biopsies usually not Wet hair or skin smears treated
worm) Microsporum lesions associated with alopecia necessary; if performed, with KOH or calcofluor white reveal
on the scalp hyphae may be visible hyphae; culture on selective agar
in biopsy material that contains cycloheximide
Tinea barbae Trichophyton Pustular lesions in bearded areas Skin biopsies usually not Wet hair or skin smears treated
verrucosum necessary; if performed, with KOH or calcofluor white
hyphae may be visible reveal hyphae; culture on selective
in biopsy material agar that contains cycloheximide
Tinea corporis (body Epidermophyton, Sharply demarcated skin lesions Skin biopsies usually not Wet hair or skin smears treated
ring worm) Microsporum, or on trunk and/or legs that contain necessary; if performed, with KOH or calcofluor white
Trichophyton pustules or papules and have hyphae may be visible reveal hyphae; culture on selective
prominent edges in biopsy material agar that contains cycloheximide
Tinea cruris (“jock itch”) Trichophyton Localized rash with scaly lesions Skin biopsies usually not Wet hair or skin smears treated
rubrum or that involve anterior aspect of necessary; if performed, with KOH or calcofluor white
Epidermophyton thighs; pustules and papules hyphae may be visible reveal hyphae; culture on selective
floccosum may be present in biopsy material agar that contains cycloheximide
Tinea manum Trichophyton Dry infection of the palmar Skin biopsies usually not Wet hair or skin smears treated
rubrum surface of the hand necessary; if performed, with KOH or calcofluor white
hyphae may be visible reveal hyphae; culture on selective
in biopsy material agar that contains cycloheximide
Tinea pedis (athlete’s T. rubrum, Pruritic foot lesions that may Skin biopsies usually not Wet hair or skin smears treated
foot) Trichophyton peel and crack and form vesicles necessary; if performed, with KOH or calcofluor white
mentagrophytes, or or pustules hyphae may be visible reveal hyphae; culture on selective
E. floccosum in biopsy material agar that contains cycloheximide
Chronic Candida albicans Uncommon disorder caused by Persistent circumscribed Demonstration of yeast and
mucocutaneous most common selective inability of T lymphocytes hyperkeratotic skin pseudohyphae on KOH smear
candidiasis to respond to Candida antigens; lesions, crumbling with confirmation by culture;
plaque-like patches with dystrophic nails; yeast identification of Candida species
erythematous borders and pseudohyphae requires biochemical tests
Sporotrichosis (usually Sporothrix schenkii Papulonodular, erythematous Skin biopsies of Skin lesions may be cultured;
involves cutaneous lesions in distal extremities; involved lesions reveal a blood cultures may be positive
and subcutaneous secondary lesions along granulomatous response if sporotrichosis is multifocal
tissues and adjacent lymphatic channels and, in some cases, cigar-
lymphatics) shaped yeast forms
Mycetoma (madura Madurella, Foot or hand infection that Hyphae may be visible Causative species inferred from
foot) Pseudallescheria extends from skin into deeper in tissue or drainage organisms in sinus tract drainage
boydii, other tissue; indurated swelling and using various stains;
species multiple sinus tracts draining pus deep biopsies are
that contains aggregates of the preferred
fungus causing the disease
Chromomycosis Fonsecaea Verrucous, cauliflower-like skin Sclerotic bodies may be Sclerotic bodies may be visible
(also known as pedrosoi; other lesions, often pruritic; may visible in stained tissue in exudates; culture confirmation
chromoblastomycosis species result in secondary infection recommended with Sabouraud
and many other names) or lymphedema agar
Skin infections caused Mycobacterium Furunculosis, wound infections, Granulomatous Isolation of organism usually
by rapidly growing fortuitum complex injection site abscesses; failure inflammation; AFB requires procedures and growth
mycobacteria and M. abscessus are to respond to commonly used stains are often negative media specific for mycobacteria
the most common antibiotics
Mycobacterium M. marinum Chronic cutaneous lesions, usually Granulomatous Isolation of M. marinum requires
marinum infection on hands, secondary to exposure to inflammation; AFB incubation of mycobacterial
marine environments or fish tanks stains are often negative media at 30°C
AFB, acid-fast bacilli; KOH, potassium hydroxide.
TABLE 5–18 Evaluation for Varicella Zoster Virus (VZV) Infection

Viral culture Vesicular fluid is the sample; the results are typically available within
7-21 days
Direct immunofluorescence assay Cells at the base of a vesicular lesion are scraped from the skin and
applied to a slide for direct immunofluorescent staining for VZV; the
detection of VZV from any source is always clinically significant as no
asymptomatic shedding of this virus is known to occur
Antibody detection assays (include These tests are used to confirm immunity to VZV, which may be
enzyme immunoassay, fluorescent important to know before and during pregnancy because a number
antimembrane antibody assay, latex of fetal anomalies are associated with VZV infection during pregnancy;
agglutination assay, and complement the anomalies depend largely on the gestational age of the fetus at
fixation test) the time of infection
PCR analysis Amplification of VZV DNA from a CSF specimen supports the diagnosis
of VZV encephalitis
Diagnosis
Chickenpox and herpes zoster are diagnosed clinically in most cases because of the character-
istic presentation of the diseases. Laboratory tests for the VZV virus, although they represent
the gold standard for diagnosis, are typically unnecessary. Cutaneous lesions may be evaluated
for the presence of VZV by direct immunofluorescence. Serologic testing is often important to
determine whether an individual has ever been infected with VZV. Assays for VZV infection are
summarized in Table 5–18.
Measles (Rubeola) and Rubella

Description
Rubeola and rubella infections are often confused because of the similarity in their names and
similar clinical manifestations. Measles (or rubeola) is a highly contagious childhood disease
characterized primarily by fever and a rash. The primary portal of entry for the rubeola virus is
the upper respiratory tract. Approximately 14 days after exposure to the rubeola virus, a charac-
teristic measles rash appears, and within 1 to 2 additional days there is a measurable amount of Rubeola and rubella
antibody to rubeola virus in the bloodstream. The leading cause of death in patients with measles infections are often
is secondary bacterial pneumonia. Rubella, also known as German measles, is most often a mild confused because of
illness in children and young adults. It is widely recognized as a dangerous infection in early preg- the similarity in their
nancy when an infection of the fetus can cause congenital abnormalities. Rubella is characterized names and similar clinical
by a rash and an enlargement of lymph nodes. Like the rubeola virus, the portal of entry of rubella manifestations. Measles
virus is most often the respiratory tract. The availability of vaccines against measles and rubella (or rubeola) is a highly
has greatly decreased the incidence of these infections in the United States. contagious childhood
disease characterized
primarily by fever and a
Diagnosis rash. Rubella, also known
The laboratory diagnosis of measles or rubella can be important for epidemiologic surveillance as German measles, is
purposes and to limit its transmission to susceptible individuals. The laboratory diagnosis is usu- most often a mild illness
ally based on detecting virus-specific IgM in serum or isolation of the virus from urine or respira- in children and young
tory specimens. adults.
Within 24 to 48 hours of the development of a rash, antibodies to rubella become detect-
able. Primary infection stimulates production of antibodies that confer lifelong immunity. It is
for this reason that the presence of antibodies to rubella is desirable before initiating pregnancy.
The antibodies can be demonstrated in a serologic test that indicates exposure to the rubella
virus. Serologic testing is an important component of the evaluation of a pregnant woman with
the clinical signs and symptoms of rubella. Table 5–19 summarizes the laboratory evaluation for
rubeola and rubella.
TABLE 5–19 Laboratory Evaluation for Measles and Rubella (German Measles)
Serology Virus-specific IgM is detectable in serum a few days after appearance of rash;
seroconversion or 4-fold rise in IgG in convalescent serum also supports the
diagnosis; presence of IgG provides evidence of immunity
Culture Specimens suitable for culture include nasopharyngeal secretions, urine, blood,
or other sterile sites; these viruses can be difficult to grow
EYE INFECTIONS
Infectious agents play a prominent role in many diseases of the eye. Table 5–20 describes the
infections of the eye according to anatomic site of infection within the eye. Many organisms that
produce eye infections are described in detail in other sections of this chapter. The microbiologic
isolation and tests for identification of the organisms listed in Table 5–20 as causative for disease
are presented in other sections of this chapter.
TABLE 5–20 Infections of the Eye and Causative Organisms

Infection Clinical Features/Definition Causative Organisms
Eyelid infections
Hordeolum An acute infection of either a meibomian gland or a gland of Zeis, Staphylococcus aureus
also known as a sty
Chalazion A chronic granuloma on a meibomian gland S. aureus
Marginal blepharitis Diffuse inflammation of the eyelid margins S. aureus has been implicated
Infections of the lacrimal system

Dacryoadenitis Inflammation of the lacrimal gland S. aureus most common; next most common
is Chlamydia trachomatis, and rarely Neisseria
gonorrhoeae
Canaliculitis An inflammation of the canaliculi Actinomyces israelii
Dacryocystitis An infection of the lacrimal system occurring as a result of outflow Acute: S. aureus, Streptococcus pyogenes, Streptococcus
obstruction in the nasolacrimal duct pneumoniae in infants, Haemophilus spp. in children.
Chronic: Actinomyces, Aspergillus, and Candida
Conjunctivitis Infection of the conjunctiva; a very common ocular infection
Viral More common than bacterial conjunctivitis in developed countries Adenovirus is the most common virus, with herpes
simplex virus, influenza A virus, enterovirus 70, and
coxsackievirus as other causative agents
Bacterial Hyperacute bacterial conjunctivitis is the most severe form Hyperacute: N. gonorrhoeae common, but also
(nonchlamydial) of conjunctivitis N. meningitidis and Corynebacterium diphtheriae.
Acute: S. aureus, S. pneumoniae, Haemophilus
influenzae in children, S. pyogenes, and Haemophilus
aegyptius; gram-negative bacillary infections are rare.
Chronic: S. aureus is the most common agent with
Moraxella lacunata and Moraxella catarrhalis also
causative
Chlamydial Two distinct presentations exist—C. trachomatis infection is a See the section “Chlamydial Infections”
leading cause of blindness in endemic areas of the world while
Chlamydia-induced inclusion conjunctivitis is a relatively benign
infection

TABLE 5–20 Infections of the Eye and Causative Organisms (continued)

Infection Clinical Features/Definition Causative Organisms
Infectious keratitis Infection of the cornea; can lead to loss of vision because
of corneal scarring or because of progression to perforation
and endophthalmitis
Viral Keratitis is almost always unilateral and may affect any age group Herpes simplex virus is the most common cause
of corneal ulcers in the United States
Bacterial Bacteria causing conjunctivitis may invade the cornea following Coagulase-negative staphylococci, S. aureus,
minor trauma to the corneal epithelium; contact lens wear is a Pseudomonas aeruginosa, S. pneumoniae, and
predisposing factor for bacterial keratitis viridans streptococci
Fungal This is a rare entity accounting for less than 2% of infectious keratitis Aspergillus, Candida, and Fusarium are the most
cases common, with geographic variation
Parasitic Most cases are in contact lens wearers Acanthamoeba is the most common cause of parasitic
keratitis in industrialized countries
Endophthalmitis Infection of the vitreous; a severe ocular infection with significant

permanent impairment of vision as a result of the infection
Postoperative This occurs in most patients 1-3 days after cataract surgery Coagulase-negative staphylococci, S. aureus, gram-
negative bacilli, streptococci, and H. influenzae
Posttraumatic The onset is rapid for virulent bacteria; onset is over weeks to Coagulase-negative staphylococci, Bacillus, gram-
months for fungal organisms negative bacilli, and fungi
Endogenous This form of endophthalmitis does not follow surgery or trauma; S. aureus, streptococci, gram-negative bacilli, Candida
usually a complication of bacteremia or fungemia
Uveitis Infection of the iris, ciliary body, and choroid (often with retinal
involvement)
Anterior Presents with redness, eye pain, photophobia; most cases of
anterior uveitis have a noninfectious immune-mediated etiology
Viral Herpes simplex 1 virus, varicella zoster virus,

cytomegalovirus
Bacterial Usually bilateral when associated with secondary syphilis Treponema pallidum (syphilis)
Posterior Visual impairment is main symptom
Viral Herpes simplex virus and the varicella zoster virus can
cause acute retinal necrosis; cytomegalovirus retinitis
mainly occurs in patients with AIDS
Bacterial T. pallidum (syphilis) is rare; perform serologic tests

for syphilis; also test CSF; Mycobacterium tuberculosis
also rare
Fungal Candida, Cryptococcus, Histoplasma
Parasitic Toxoplasma gondii is a common cause;

Toxocara canis mainly affects children
INFECTIONS OF THE LARYNX, PHARYNX,

MOUTH, EAR, ORBIT, AND SINUSES
Table 5–21 describes the infections of the pharynx, larynx, mouth, ear, orbit, and sinuses. Because
there are such a large number of organisms that produce these infections, a brief description of
the infections and their associated organisms is presented in Table 5–21. For similar reasons, the
diagnostic studies useful in identifying the disease and its causative organisms are also provided
in the table.
TABLE 5–21 Infections of the Larynx, Pharynx, Mouth, Ear, Orbit, and Sinuses
Histopathology/
Disease or Pathogen Clinical Findings Radiology Microbiology Testing Common Pathogens
Laryngeal infections
Laryngitis, acute Hoarseness and Histopathologic and Diagnosis usually on clinical Influenza viruses,
occasional aphonia are radiographic studies are features rhinoviruses, adenovirus,
associated with upper not useful for routine parainfluenza viruses,
respiratory infections diagnosis Streptococcus pneumoniae,
Haemophilus influenzae, and
Streptococcus pyogenes
Laryngitis, Cough, wheezing, Granulomatous changes Laryngeal biopsy may be Mycobacterium tuberculosis
tuberculous (laryngeal hemoptysis, dysphagia, and acid-fast organisms submitted for mycobacterial (highly infectious)
tuberculosis) (also odynophagia; laryngeal may be observed in smears and culture; blood
see the section lesions vary from ulcers laryngeal biopsy material; cultures may be positive for
“Tuberculosis”) to exophytic masses chest radiographs mycobacteria
may reveal pulmonary
tuberculosis
Pharyngeal and oral infections
Herpes Painful, ulcerating Rapid diagnosis with DFA stain; rapid antigen Herpes simplex virus is the
gingivostomatitis vesicles in oral mucosa; Giemsa- or Wright-stained detection of moist lesion agent of primary infection
fever, fetid breath, smears from lesion by scrapings may be positive; can
cervical adenopathy, identifying multinucleated provide rapid diagnosis; less
drooling; usually in giant cells (less sensitive sensitive than culture; lesions
children less than than culture); Tzanck may be cultured, usually for
5 years old preparation; less sensitive 24-48 h; PCR available in some
than DFA culture labs (more sensitive than
culture); serologic tests of
acute and convalescent sera
may aid in diagnosis
Herpes labialis, Painful, ulcerating Rapid diagnosis with DFA stain; rapid antigen Herpes simplex virus is the
recurrent vesicles beginning on the Giemsa- or Wright-stained detection in moist lesion agent of recurrent disease
outer lip (usually lower smears from lesion by scrapings may be positive; can
lip); fever usually absent identifying multinucleated provide rapid diagnosis; less
giant cells (Tzanck sensitive than culture; lesions
preparation); less sensitive may be cultured, usually
than DFA or culture for 24-48 h; PCR available in
some labs (more sensitive
than culture); serologic tests
generally not useful
Oral thrush (oral Creamy white patches Histopathologic studies KOH or Gram-stained Candida albicans
candidiasis) on the tongue and oral are not useful for routine smears of oral lesions reveal
mucosa that bleed easily diagnosis pseudohyphae and yeast
when scraped forms
Streptococcal Pharyngeal pain, Histopathologic studies Rapid antigen detection by S. pyogenes (group A
pharyngitis (“strep odynophagia, fever, are not useful for routine throat swab (less sensitive streptococci); groups C and
throat”) chills, headache; anterior diagnosis than culture); routine culture G streptococci cause milder
cervical adenopathy; of throat (posterior pharynx) pharyngitis; infections of the
purulent exudates, swab is most sensitive throat by respiratory viruses
edema, and erythema may mimic strep throat
in posterior pharynx clinically
Neisseria gonorrhoeae infection of the pharynx (see the section “Gonorrhea”)
Diphtheria In its mildest form, Histopathologic studies Organisms from lesion can be Corynebacterium diphtheriae
asymptomatic carriage are not useful for routine grown in culture (specialized
of organisms; formation diagnosis agar improves sensitivity)
of tough membrane over
pharyngeal surface; also
can cause skin lesions
and damage to multiple
organs

TABLE 5–21 Infections of the Larynx, Pharynx, Mouth, Ear, Orbit, and Sinuses (continued)
Histopathology/
Disease or Pathogen Clinical Findings Radiology Microbiology Testing Common Pathogens
The “common cold” Nasal discharge and Not useful Testing to rule out a bacterial Rhinovirus, coronavirus, and
sinus congestion; often infection, usually by culture of a adenovirus, among others
with pharyngeal and throat swab specimen, is often
sinus pain; may be febrile performed when pharyngeal
with chills and headache pain is present; multiplex
nucleic acid amplification tests
may be useful in patients with
underlying risk factors
Ear, orbit, and sinus infections
Otitis externa Pruritic and painful outer If invasive otitis externa Wound or external auditory Pseudomonas aeruginosa
ear, with an edematous present, CT or MRI of head canal specimens for Gram stain (swimmer’s ear),
and erythematous ear is useful for monitoring and culture Staphylococcus aureus, and
canal bone or tissue infection S. pyogenes
Otitis media Ear pain, otorrhea, hearing Histopathologic and Diagnosis usually on clinical S. pneumoniae, H. influenzae,
loss with fever, irritability, radiographic studies are features; tympanic fluid Moraxella catarrhalis,
headache, lethargy, not useful for routine obtained by tympanocentesis S. pyogenes, S. aureus,
anorexia, and vomiting diagnosis may be cultured and selected viruses
Orbital cellulitis Proptosis and eye pain; Cranial CT scan of sinuses Blood, conjunctival, and S. aureus, S. pyogenes,
eyelid swelling, redness, and orbit may identify wound specimens for Gram H. influenzae,
warmth, tenderness abscesses stain and culture and S. pneumoniae
Periorbital cellulitis Eyelid pain, swelling, and Sinus radiographs or CT Blood, conjunctival, and H. influenzae and anaerobic
erythema with low-grade scan may exclude sinus wound specimens for Gram bacteria
fever disease stain and culture
Sinusitis (acute) Persistent upper For complicated cases, Usually a clinical diagnosis; S. pneumoniae, H. influenzae,
respiratory symptoms; CT scanning of paranasal sinus puncture aspirates are and rhinoviruses
purulent nasal discharge, sinuses is method of specimen of choice for Gram
fever, facial pressure or choice—presence of an stain and culture; endoscopic
pain, facial erythema air-fluid level correlates sampling of exudates less likely
or swelling, and nasal with bacterial infection to identify pathogens
obstruction
CT, computed tomography; KOH, potassium hydroxide; MRI, magnetic resonance imaging.
INFECTIONS OF THE LUNG AND PLEURAE It is very important to

consider the clinical
Overview setting when evaluating
Many categories of organisms can cause pneumonia and other types of pulmonary infections. and managing a patient
It is very important to consider the clinical setting when evaluating and managing a patient with with pneumonia because
pneumonia because the types of organisms that are responsible depend on whether or not specific the types of organisms
that are responsible
risk factors are present.
depend on whether or
Community-acquired pneumonia is commonly caused by S. pneumoniae, Mycoplasma
not specific risk factors
pneumoniae, respiratory viruses, H. influenzae, and Legionella spp. In contrast, hospital-associated and
are present.
ventilator infections are likely to be caused by multidrug-resistant organisms including K. pneumoniae,
P. aeruginosa, Acinetobacter baumanii complex, and MRSA. P. aeruginosa, Burkholderia cepacia, and
MRSA are also important causes of lung infections in patients with cystic fibrosis. In recent years
there has been an increase in the number of Bordetella pertussis infections that has been attributed to
waning immunity following the introduction of acellular pertussis vaccines.
Travel and/or exposure history can be an important clue in patients with persistent pulmo-
nary signs and symptoms, as they may have TB or dimorphic fungal infections (described below).
Patients with depressed cell-mediated immunity (eg, transplant recipients or HIV infection) are
at increased risk of Pneumocystis and CMV infections. Patients who have prolonged neutropenia
from chemotherapy, for example, are at increased risk of invasive infections caused by Aspergillus
spp. and other fungi.
Table 5–22 describes the infections of the lung and pleurae. The many different lung infec-
tions are grouped into bacterial, fungal, parasitic, and viral diseases. There are 2 major challenges
TABLE 5–22 Infections of the Lung and Respiratory Tract

Histopathology
Pathogen Clinical Findings and Radiology Microbiology Testing Other Tests
Bacterial infections
Bordetella pertussis Paroxysmal, nonproductive CXR may show Cultures and/or PCR Peripheral blood
(whooping cough) cough; low-grade fever, pneumonia with from nasopharyngeal lymphocytosis often present
rhinorrhea; vomiting may consolidation specimens
follow cough
Burkholderia cepacia Causes respiratory distress or CXR may show Sputum from lower
progressive respiratory failure widespread infiltrates respiratory tract for Gram
with high fever in cystic fibrosis stain and culture with
patients (especially females) special media
Moraxella catarrhalis Tracheitis, bronchitis, sinusitis, In most cases, CXR Gram stain and culture Serologic tests not useful
and otitis media can all occur findings are not from respiratory
prominent specimens
Chlamydia Pharyngitis, hoarseness, fever, CXR usually reveals Chlamydia culture possible IgM and IgG serologic tests for
pneumoniae mild pneumonitis; atypical single subsegmental but requires specialized antibody to the organism may
pneumonia, especially in the lesion; pleural effusion cell lines; PCR assays be useful for diagnosis; PCR
elderly may be evident for direct detection are assays for direct detection are
commercially available commercially available
Coxiella burnetii Causes atypical pneumonia CXR often shows Culture and PCR only IgM and IgG serologic tests
(Q fever) with fever, severe headache, multiple rounded performed in specialized for antibody to the organism
chills, sweats, myalgias; opacities or reference laboratories most useful for initial
associated with exposure diagnosis; normal WBC count;
to livestock elevated smooth muscle
autoantibodies often present
Klebsiella Bronchitis, bronchopneumonia, CXR may reveal pattern Sputum from lower
pneumoniae or lobar pneumonia; “currant of pneumonia and respiratory tract for Gram
jelly” sputum; frequent identify complications, stain and culture
complications such as abscess if they arise
and empyema
Haemophilus Pneumonia with fever, CXR may show interstitial Sputum or other lower
influenzae productive cough; often or bronchopneumonia, respiratory tract specimen
(nontypeable) exacerbates chronic bronchitis or pneumonia with for Gram stain and culture
consolidation
Legionella Atypical pneumonia with CXR typically shows Respiratory specimens Hyponatremia often present;
pneumophila slightly productive cough, alveolar infiltrates; cultured on selective PCR and serologic tests may
(also see the fever, and chest pain; diarrhea pleural effusions media; urinary antigen test be useful for diagnosis
section “Legionella often present common is rapid and sensitive for
Infections”) serogroup 1
Mycobacterium In the non-HIV-infected CXR mimics reactivation Sputum from lower In HIV-infected population,
avium-intracellulare patient, pulmonary disease tuberculosis with respiratory tract specimen must distinguish atypical
complex (another with productive cough, fever, cavitation for acid-fast stain and mycobacteria from
nontuberculous weight loss, and occasionally culture (must distinguish Mycobacterium tuberculosis
mycobacterium) hemoptysis active disease from infection
colonization)
M. tuberculosis (see the section “Tuberculosis”)
Mycoplasma Often causes an upper CXR may show Difficult to diagnose by IgM and IgG tests are useful
pneumoniae respiratory tract infection with extensive infiltrates, microbiologic culture; (may require acute and
fever, malaise, headache, and out of proportion with cultures require 1-4 weeks convalescent serum); can
nonproductive cough; may symptoms for growth with special also be used for diagnosis
cause atypical pneumonia media; not detected by
Gram stain
Pseudomonas Causes pneumonia in elderly, CXR may reveal diffuse Sputum from lower Mucoid isolates often
aeruginosa hospitalized, and cystic fibrosis bronchopneumonia; in respiratory tract for Gram obtained from cystic fibrosis
patients; may be fulminant with bacteremic pneumonia, stain and culture; blood patients
chills, fever, dyspnea, excessive alveolar and interstitial cultures may be positive
purulent sputum, and cyanosis infiltrates with cavitation
may be seen

TABLE 5–22 Infections of the Lung and Respiratory Tract (continued)

Histopathology
Staphylococcus Pneumonia with fever, CXR shows infiltrates, Sputum from lower Empyema is a frequent
aureus purulent sputum consolidation, abscesses, respiratory tract for Gram complication that requires
pleural effusions, and/or stain and culture; pleural drainage; pleural fluid
loculations fluid or empyema if present very purulent with many
should be cultured; blood neutrophils
cultures may be positive
Streptococcus Causes pneumonia in neonates CXR in neonates may Sputum from lower Pregnant carrier females
agalactiae (group B and elderly; fever present; show pulmonary respiratory tract for Gram may be screened by culture
streptococci) apnea, tachypnea, grunting, infiltrates, often similar stain and culture of vaginal and rectal swab
and cyanosis in neonates to hyaline membrane specimens; nucleic acid
disease amplification tests are an
alternative
Streptococcus Productive cough with rust- CXR may show Sputum from lower Peripheral blood leukocytosis
pneumoniae tinged sputum, fever, shaking subsegmental respiratory tract for frequent; empyema is an
chills, and pleuritic chest pain infiltrations; segmental Gram stain and culture; uncommon complication
or lobar consolidation blood cultures may yield
may be present organisms
Streptococcus Abrupt onset of pneumonia CXR reveals Sputum from lower Empyema is a frequent
pyogenes (group A with fever, chills, dyspnea, bronchopneumonia with respiratory tract for Gram complication
streptococci) pleurisy, and blood-tinged consolidation stain and culture; blood
sputum cultures may be positive
Fungal infections (see the section “Dimorphic Fungi and Other Fungal Infections”)
Pneumocystis (see the section “Pneumocystis jirovecii Pneumonia”)

Viral infections
Adenovirus Pharyngitis or tracheitis with Adenoviral eosinophilic Adenoviral culture from With serologic testing, a
cough, fever, sore throat, inclusions may be visible respiratory specimens; 4-fold rise in titer is consistent
and rhinorrhea; interstitial in lung biopsies if they rapid viral antigen with new infection
pneumonia may develop; are obtained detection by DFA can
diarrhea also may be present be useful; nucleic acid
amplification is useful
Cytomegalovirus Interstitial pneumonitis with CXR shows interstitial Viral cultures from Quantitative PCR assays
(CMV) nonproductive cough, fever, pneumonia; nodules or respiratory specimens using blood are preferred for
dyspnea, and hypoxia cavities may be seen; (bronchoalveolar lavage diagnosis of disseminated
lung biopsies reveal CMV fluid or tissue); can also disease
inclusions (“owl’s eye” use PCR
cells)
Hantavirus Fever, severe myalgias, CXR shows rapid Immunohistochemistry IgM serologic tests by capture
headache, tachypnea, and progression to bilateral or PCR using blood or enzyme immunoassay or
shortness of breath; rapidly interstitial edema and lung biopsy may confirm Western blot are diagnostic
progressive to hypotension, diffuse alveolar disease; infection methods of choice
respiratory failure, and shock pleural effusions often
present
Influenza A or B virus Fever, chills, myalgias, CXR may show bilateral Nucleic acid amplification Rapid antigen detection tests
headaches, dry cough; primary infiltrates (RT-PCR) performed have poor sensitivity, often
viral pneumonia may occur on nasopharyngeal less than 50%
specimens is most
sensitive method; DFA plus
viral culture is also useful
Human Bronchiolitis and pneumonia CXR may show Nucleic acid amplification
metapneumovirus similar to respiratory syncytial interstitial infiltrates or (RT-PCR) performed on
virus infections hyperinflation nasopharyngeal specimens
is most sensitive method;
DFA test is less sensitive;
grows poorly in culture

TABLE 5–22 Infections of the Lung and Respiratory Tract (continued)

Histopathology
Parainfluenza viruses Upper respiratory tract CXR is negative in cases Nucleic acid amplification
1, 2, 3, and 4 infections, otitis media, with no pulmonary (RT-PCR) performed
conjunctivitis, and pharyngitis; involvement on nasopharyngeal
may cause croup or specimens is most
bronchiolitis sensitive method; DFA plus
Respiratory syncytial Pneumonia or bronchiolitis; CXR may show Nucleic acid amplification Rapid antigen detection tests
virus (usually infants fever, paroxysmal cough, interstitial infiltrates or (RT-PCR) performed have moderate sensitivity
and young children) dyspnea hyperinflation on nasopharyngeal (50%-80%)
specimens is most
sensitive method; DFA plus
Pleural empyema: Chest pain, chills, persistent Thoracic CT scan usually Pleural or empyema fluid Peripheral blood leukocytosis
most common fever, right sweats permits definitive should be cultured for usually present; pleural fluid
organisms found are diagnosis; ultrasound aerobic and anaerobic values often show fluid pH
S. pneumoniae, S. distinguishes solid organisms below 7, glucose below 40
aureus, H. influenzae, lesions from pleural mg/dL, and LDH exceeding
S. pyogenes, P. fluid collections; CXR 1000 IU/L
aeruginosa, K. may show pleural fluid
pneumoniae, and accumulations in lateral
Bacteroides decubitus views
CXR, chest radiograph; LDH, lactate dehydrogenase; PCR, polymerase chain reaction; CT, computed tomography.
in the laboratory diagnosis of pulmonary infections. First, it can be difficult to obtain respira-
tory specimens that are not contaminated with oropharyngeal flora. This is particularly true
of expectorated sputum. It is 1 of the reasons why this type of specimen is routinely screened
microscopically for the presence of squamous epithelial cells to determine whether it is a true
lower respiratory specimen. The other problem is that there is no single test that detects all of the
potential respiratory pathogens. While routine Gram stain and sputum culture readily detects
S. pneumoniae, common gram-negative rods, and S. aureus, separate cultures and/or test methods
are required to detect Legionella, mycobacteria, fungi, respiratory viruses, CMV, and Pneumocys-
tis. These are discussed in more detail in the following sections.
Tuberculosis
Description
TB is a major cause of morbidity and mortality around the world and remains a major chal-
Approximately one third
lenge for public health officials. Approximately one third of the world’s population is estimated
of the world’s population
to be infected with the causative agent, M. tuberculosis. This slow-growing AFB continues to be
is estimated to be infected
with the causative agent,
an important concern in industrialized countries because of the development of drug-resistant
M. tuberculosis. Early strains and a growing population of immunosuppressed patients who are at increased risk of TB.
identification of patients Although it can infect a variety of organs, M. tuberculosis primarily causes pulmonary disease.
with active pulmonary TB It is usually acquired by inhalation of infectious aerosolized droplets. The majority of primary
is crucial for preventing infections are asymptomatic; however, the organisms are not completely eliminated. This leads
transmission of this to a quiescent phase known as latent tuberculosis infection (LTBI). Otherwise healthy individu-
serious infection to other als with LTBI have an approximately 10% lifetime risk of developing secondary or reactivation
patients and to health pulmonary TB. Prophylaxis of asymptomatic-infected individuals reduces the risk of subsequent
care workers. reactivation TB.
Clinical features of active pulmonary TB include fever, night sweats, weight loss, productive
cough, and hemoptysis in later stages of the disease. Radiographic studies often show cavitary
lung lesions, usually in the lung apices. It is important to realize that immunosuppressed patients
who develop active TB often have atypical clinical presentations and can have nonspecific radio-
graphic changes. Before the epidemic of HIV infection in the United States, approximately 85%
of newly diagnosed infections with TB were limited to the lung, with 15% involving nonpulmo-
nary sites or both pulmonary and nonpulmonary sites. With advanced HIV infection, less than
half the cases are limited to pulmonary involvement. Extrapulmonary TB commonly involves
the lymph nodes, pleura, genitourinary tract, bones and joints, meninges, peritoneum, and peri-
cardium.
Early identification of patients with active pulmonary TB is crucial for preventing transmis-
sion of this serious infection to other patients and health care workers. Treatment of active TB
caused by sensitive strains requires combination therapy for 6 months. Multidrug-resistant and
extremely resistant M. tuberculosis (MDR-TB and XDR-TB, respectively) are more difficult to
treat and have a poorer outcome.
Diagnosis
There are 2 categories of laboratory tests for TB: those that detect latent infection and those
that detect active disease. Skin testing with PPD detects previous exposure to M. tuberculosis.
A delayed-type hypersensitivity response to the M. tuberculosis antigens (mediated by T cells)
leads to induration at the site of injection. One problem with the PPD test is that individuals who
have been vaccinated with bacille Calmette–Guerin (BCG) can also have a positive reaction. BCG
is derived from M. bovis, a member of the M. tuberculosis complex, and is widely used outside
the United States. Recent advances in immunology and genomics have led to the development of
alternatives to the PPD test, such as interferon-gamma release assays (IGRAs). Peripheral blood
or purified mononuclear cells are incubated with antigenic peptides that are unique to M. tuber-
culosis (ie, they are not present in BCG) and then an immunoassay is performed to measure pro-
duction of interferon-gamma. The IGRAs are at least as sensitive as the PPD and are more specific
since BCG vaccination does not produce a false-positive result.
The diagnosis of secondary TB depends on detection of M. tuberculosis in clinical samples.
This testing is particularly important since the PPD and IGRAs can be negative in patients with
active TB. Acid-fast staining of sputum specimens (using either a fuchsin [red] stain or a fluores-
cent stain) enables visualization of the mycobacteria in ~70% of cases of pulmonary TB. Detection
of mycobacteria in sputum using an AFB stain provides only presumptive evidence of pulmonary
TB in the presence of characteristic radiologic findings. The presence of M. tuberculosis must be
confirmed by culturing the organism in liquid and/or solid media or by using NAATs. Modern
automated liquid culture systems routinely detect growth of M. tuberculosis in 1 to 2 weeks versus
4 to 6 weeks with traditional culture on solid media. These systems also make it possible to per-
form rapid susceptibility testing in liquid culture for first-line antituberculous drugs. The NAATs
make possible same-day confirmation of smear-positive specimens, which contain relatively large
numbers of organisms. Culture remains the gold standard for detecting M. tuberculosis in smear-
negative specimens.
Table 5–23 includes the clinical and laboratory information relevant to the diagnosis of pul-
monary and extrapulmonary TB.
Other nontuberculous mycobacteria, including slow-growing organisms such as M. avium
complex and M. kansasii, and rapid growers, such as M. abscessus, can cause chronic pulmonary
disease in both normal and immunocompromised hosts.
Legionella Infections
Description
Legionella pneumophila is a fastidious, slow-growing gram-negative rod. Legionella species
are widespread in the environment and are a cause of community-acquired pneumonia. They
are usually found in surface or potable water and are associated with moist environments.
Approximately 10,000 to 15,000 cases of Legionella pneumonia occur each year in the United
States. Most cases occur sporadically, but outbreaks have been associated with aerosolized
transmission from cooling towers, evaporative condensers, potable hot water lines, show-
ers, respiratory therapy equipment, decorative fountains, and whirlpool spas. Outbreaks in
health care facilities are especially worrisome because of the large population of patients with
compromised immunity or impaired pulmonary function who are at increased risk of severe
Legionella infections.
TABLE 5–23 Evaluation for Tuberculosis (TB)

Pulmonary Tuberculosis CNS Tuberculosis Genitourinary Tuberculosis Disseminated Tuberculosis
Clinical Symptoms range from Fever, unremitting Most common site for More likely to occur in HIV-
findings none to fever with headache, nausea, and extrapulmonary TB is the kidney; positive individuals; may
productive cough and malaise; in the United dysuria, frequency, and hematuria be present without miliary
dyspnea; hemoptysis States, elderly are are common; women may present pattern in chest radiographs;
indicates presence of frequently affected; where with a chronic pelvic inflammatory patient may present with fever,
advanced disease TB is common, it primarily process, menstrual irregularities, or weight loss, and anorexia
affects children aged sterility; men may present with an
1-5 years enlarging scrotal mass
Tests
PPD or In the presence of In the presence of In the presence of compatible In the presence of compatible
interferon- compatible radiologic and compatible radiologic and radiologic and clinical findings, a radiologic and clinical
gamma clinical findings, a positive clinical findings, a positive positive PPD in an unvaccinated findings, a positive PPD in
release assay PPD in an unvaccinated PPD in an unvaccinated patient, or a positive IGRA, an unvaccinated patient, or
(IGRA for TB) patient, or a positive IGRA, patient, or a positive IGRA, suggests TB; a negative result does a positive IGRA, suggests TB;
suggests TB; a negative suggests TB; a negative not exclude active infection a negative result does not
result does not exclude result does not exclude exclude active infection
active infection active infection
Microscopy Acid-fast bacilli in sputum Acid-fast bacilli in smears Both urine and sputum samples Urine, lymph node, liver,
smears permit rapid of CSF lead to identification should be examined, as a smear bone marrow, and sputum
diagnosis; sensitivity is of 20% or less of CNS TB from a urine sample may not have smears have low sensitivity
variable, but increases cases; sputum samples also detectable acid-fast bacilli for organism detection
with the number of should be tested
specimens examined (up
to 4)
Mycobacterial Culture from sputum Culture of CSF may reveal Urine specimens for mycobacterial Culture may be performed
culture specimen on liquid and organisms in CNS TB cases culture are positive in 60%-80% of using bone marrow, liver,
solid media is the most cases, although it is more likely to urine, and sputum specimens
sensitive method; for be positive in men than in women
pediatric cases, multiple
gastric lavage specimens
can be used; liquid
culture with DNA probe
hybridization enables
rapid TB confirmation
Nucleic acid Very useful for rapid May provide a rapid Utility not well defined Sputum specimens may be
amplification detection of TB but does diagnosis but cannot used for amplification
not replace culture replace culture
Other findings Pleural fluid, if present, With lumbar puncture, In the appropriate clinical setting, Impaired function of infected
is an exudate (not there may be an increased TB may be considered if negative organs may be noted in
a transudate) with opening pressure and 100- routine urine cultures show WBCs routine laboratory tests
mononuclear cells 1000 cells/μL of CSF (mostly in acid urine of those organ systems
mononuclear cells) and
elevated CSF protein
Radiology Chest radiograph may If TB is established in the 40%-75% of cases have a positive Chest radiograph may be
detect adenopathy, brain, it may produce a chest radiograph; other radiologic normal and repeat testing may
effusion, cavitation, or mass, or “tuberculoma,” studies are not very useful prove useful; CT scan or MRI
nodule; in HIV-infected visible by CT scan may be useful to detect TB in
patients, the chest extrapulmonary sites such as
radiograph is less likely the brain or vertebrae
to show typical changes
Anatomic Caseating granulomas Biopsy may be diagnostic Renal biopsy may be helpful to If bronchial washings do not
pathology may be observed in identify genitourinary lesions provide diagnosis, granulomas
biopsies of enlarged in bone marrow or liver biopsy
lymph nodes may be diagnostic
CNS, central nervous system; CSF, cerebrospinal fluid; CT, computed tomography; MRI, magnetic resonance imaging; PPD, purified protein derivative.
Legionella infections can present as subclinical infections, pneumonia, and extrapulmonary

infections including endocarditis. Patients with Legionella pneumonia can present with a broad
spectrum of symptoms, ranging from mild cough to widespread pulmonary infiltrates and mul-
tisystem failure. Patients with Legionella pneumonia may also experience hemoptysis, diarrhea,
and a change in mental status.
Diagnosis
The diagnosis of legionellosis can be easily missed because the organisms are not detected
on routine Gram stain (Legionella stains very poorly) and growth of the organism in culture
requires special types of agar. A sputum Gram stain showing mostly neutrophils, without asso-
ciated bacteria, should raise suspicion of Legionnaires disease or other atypical pneumonia.
L. pneumophila serogroup 1 infections, which account for most community-acquired legio-
nellosis, can be readily diagnosed using rapid immunoassays that detect a Legionella antigen
that is excreted in urine. These tests detect 80% to 90% of cases and have good specificity.
Another advantage is that they remain positive for several weeks, even after the patient has
been started on antibiotics. Bacterial culture is the gold standard for the diagnosis of Legionella
infection. Cultures from sputum, bronchoalveolar lavage (BAL), and/or lung tissue may require
4 to 5 days of growth for isolation of Legionella colonies. The sensitivity for organism detec-
tion is greater with a BAL specimen than with an expectorated sputum specimen. Isolation of
Legionella from specimens requires the use of a charcoal-based bacteriologic medium, with
the addition of antibiotics if the specimens are from nonsterile sites. The isolation and identi-
fication of Legionella, in association with pneumonia, is diagnostic for Legionella pneumonia.
Direct fluorescent antibody tests of respiratory specimens are no longer recommended because
they are relatively insensitive, and the specificity is greatly dependent on the experience of the
individual performing the test. PCR testing and serology also may be useful in diagnosis of
Legionella infections.
Table 5–24 summarizes the laboratory tests relevant to diagnosis of the Legionella infections.
Nocardiosis
Description
Nocardia spp. are aerobic gram-positive actinomycetes that are found worldwide in soil and
decaying organic matter. Nocardiosis is chiefly an opportunistic infection, particularly in patients
with impaired cell-mediated immunity such as hematopoietic malignancies, HIV/AIDS, those
receiving immunosuppressive therapy, and transplant recipients. Pulmonary nocardiosis is the
most common presentation. It can exhibit the full spectrum of acute or chronic pulmonary infec-
tion, including pneumonia and abscess formation. Other clinical manifestations include anorexia,
productive cough, pleurisy, dyspnea, hemoptysis, and weight loss.
TABLE 5–24 Evaluation of Patients for Legionnaires Disease

Laboratory Test Result
Bacteriologic culture This is the “gold standard” test that requires special media for growth and isolation of
the Legionella organisms
Urinary antigen Detects only Legionella serogroup 1; overall sensitivity is 60%-80% because serogroup
1 represents 60%-80% of Legionella pneumonia cases; within serogroup 1,
the sensitivity compared with culture is ≥95%
Direct fluorescent Generally performed on colonies isolated from bacteriologic plates; less useful on
antibody test respiratory specimens and lung biopsies due to lower sensitivity and specificity
Serology Rising titers of antibody to Legionella may be useful in documentation of disease

in culture-negative cases
PCR analysis Amplification of Legionella DNA from respiratory specimens is a means to detect
all Legionella pathogens—useful in selected situations
PCR, polymerase chain reaction.
TABLE 5–25 Evaluation for Nocardiosis

Cutaneous/Subcutaneous
Pulmonary Nocardiosis Nocardiosis CNS Nocardiosis Systemic Nocardiosis
Microbiology Gram stain, modified Gram stain, modified Aerobic culture may Testing is most useful for cases
acid-fast stain, or aerobic acid-fast stain, or aerobic generate a positive test in involving CNS and lungs; blood
culture may generate culture may generate CSF specimens or aspirates culture may be positive if processed
a positive result from a positive result from of cerebral masses; Gram to maximize organism recovery;
sputum and bronchial specimens obtained from stain or modified acid- specimens from sites of suspected
specimens; selective agar fistulas, abscesses, or skin fast stain may reveal involvement may be used for
increases yield biopsies filamentous bacteria smears and aerobic cultures
Anatomic Biopsies of large cavitary Biopsies of skin lesions Fine needle aspirate of Biopsies of affected organs or
pathology lesions in the lung may may reveal organisms cerebral mass may reveal tissues may reveal organisms
reveal organisms organisms
CNS, central nervous system; CSF, cerebrospinal fluid.
Primary Nocardia infection in the lung, skin, or soft tissue may erode into blood vessels and
spread hematogenously to a variety of different organs. Nocardia have a well-recognized predilec-
tion for invasion into the CNS.
Diagnosis
Detection of the organism can be accomplished by microscopic examination of specimens com-
bined with culture. Gram staining may reveal filamentous gram-positive rods with or without
branching. They are also partially acid-fast; that is, they are positive on a modified acid-fast stain
(which uses a less stringent decolorizer) but are negative on a regular acid-fast stain. Nocardia spp.
are slowly growing organisms and may be difficult to recover. Traditionally they were identified
based on biochemical reactions. More recently introduced DNA-based methods reveal that many
of organisms that would previously have been identified as Nocardia asteroides are separate spe-
cies with distinct antibiotic susceptibility profiles.
The laboratory information for diagnosis of nocardiosis in different anatomic sites is pro-
vided in Table 5–25.
Pneumocystis jirovecii Pneumonia

Description
Pneumocystis spp. are single-cell organisms that were originally described as protozoans, but phy-
logenetic analysis indicates that they are more appropriately classified with the fungi. These organ-
isms are a well-recognized cause of pulmonary infection in patients with profoundly impaired
cell-mediated immunity. These infections were originally attributed to P. carinii (which is found
in rats), but it is now known that human infections are caused by the morphologically similar
P. jirovecii. From the beginning of the HIV epidemic in the early 1980s until 1993, P. jirovecii was
the indicator infection for more than 20,000 newly diagnosed cases of AIDS in the United States
reported to the Centers for Disease Control and Prevention. It has become much less common
since the introduction of highly active antiretroviral therapy. In a small number of cases, Pneumo-
cystis can also cause extrapulmonary infections.
Diagnosis
Pneumocystis spp. cannot be cultured in vitro. Laboratory diagnosis depends on identification
of the organism in stained preparations of clinical specimens, most often induced sputum or
The dimorphic fungi grow BAL fluid. These are concentrated onto a slide by cytocentrifugation. Other specimens include
as filamentous molds transbronchial or open lung biopsies. The Pneumocystis life cycle includes trophozoite and cyst
in the environment but stages. The most sensitive method for detecting these forms is staining the preparation with fluo-
transform into yeast (or rescently labeled monoclonal antibodies. Other frequently used stains are GMS (stains cyst walls)
related forms) in infected and Giemsa (stains trophozoites and intracystic stages). PCR analysis of respiratory specimens
tissue. may be useful in the diagnosis of Pneumocystis, but it is not widely available.
Table 5–26 summarizes the laboratory information that supports a diagnosis of P. jirovecii
infection.
TABLE 5–26 Evaluation for Pneumocystis jiroveciia

Type of Infection Specimen Laboratory Test Results/Comments
Pneumocystis BAL fluid, induced Microscopy using special stains is Fluorescently labeled monoclonal antibodies can detect cysts
pneumonia sputum, or lung biopsy the standard method for diagnosis and trophic forms. Gomori methenamine silver (GMS) stain
of Pneumocystis pneumonia only detects cyst walls; Giemsa stain only detects trophic forms
Extrapulmonary Lymph node, spleen, Microscopy using special stains Organisms can be detected with GMS or fluorescent
Pneumocystis bone marrow, or liver antibodies; they do not stain with hematoxylin and eosin
infection
a
In older literature, this organism is referred to as P. carinii (see Stringer JR, Beard CB, Miller RF. Spelling Pneumocystis jirovecii. Emerg Infect Dis. 2009;15:506).
Dimorphic Fungi and Other Fungal Infections

Description
The dimorphic fungi grow as filamentous molds in the environment but transform into yeast (or
related forms) in infected tissue. The most important members of this group are H. capsulatum
and Coccidioides spp. These organisms are usually acquired by inhalation, but they can dissemi-
nate and cause life-threatening systemic infections.
H. capsulatum is endemic along the Mississippi and Ohio River Valleys, as well as in parts
of Central America and the Caribbean region. Most infections are asymptomatic or subclinical;
however, inhalation of large quantities of spores or hyphal fragments can cause symptomatic lung
infection that requires antifungal therapy. As with TB, primary infection with H. capsulatum is
contained by the cell-mediated immune response. However, the organism may not be eradicated.
H. capsulatum infection in patients with underlying lung disease can lead to chronic progressive
pulmonary histoplasmosis that must be treated to prevent further lung damage. Patients who
have depressed cell-mediated immunity (due to underlying disease or immunosuppressive ther-
apy) are at a risk of developing disseminated histoplasmosis. This form of the disease may pres-
ent with nonspecific findings such as fever, weight loss, and hepatosplenomegaly, and it is fatal if
untreated. Patients who harbor H. capsulatum and receive drugs that inhibit TNF or its receptor
are also at a high risk of developing disseminated infection.
Coccidioides spp. are endemic in the southwestern United States (C. immitis in the central
valley of California and C. posadasii in Arizona). The life cycle of Coccidioides is similar to
H. capsulatum except that it forms spherules in infected tissue. Immunosuppressed patients
and certain ethnic groups are at increased risk of disseminated coccidioidomycosis and CNS
infections.
Immunosuppressed patients are susceptible to a variety of other fungal lung infections,
including Aspergillus spp. and other septate molds, nonseptate molds such as Mucor and Rhizo-
pus, and the encapsulated yeast C. neoformans.
Diagnosis
Diagnostic tests for H. capsulatum include fungal culture, immunoassays that detect a fungal cell
wall antigen, and serology. Culture is the gold standard, but fungal growth is usually not detected
for 1 to 2 weeks. Serology is useful for patients with chronic pulmonary histoplasmosis, but it is
relatively insensitive for diagnosis of disseminated histoplasmosis. The antigen test performed on
serum or urine is particularly useful for diagnosing disseminated disease. Coccidioidomycosis
is diagnosed by serology and/or culture depending on the clinical presentation. The diagnosis of
dimorphic fungal infections can also be confirmed by demonstration of characteristic structures
in biopsy specimens. Currently the diagnosis of other fungal lung infections relies on culturing
respiratory secretions and/or histopathologic examination of biopsy specimens. A serum assay
that detects a galactomannan antigen produced by Aspergillus spp. is useful in the diagnosis of
invasive pulmonary aspergillosis. Serum assays for circulating β-d-glucan may also be useful for
detecting invasive fungal infections.
The clinical findings associated with systemic mycotic infections are shown in Table 5–27
along with the microbiologic evaluation and histopathology findings.
TABLE 5–27 Evaluation for Systemic Mycotic Infections

Pathogen Clinical Findings Microbiology Histopathology
a
Dimorphic fungi
Blastomyces dermatitidis Chronic pneumonia with productive Broad-based budding yeasts may be visible in Broad-based budding
(occurs in parts of the cough, hemoptysis, weight loss, and calcofluor stains of wet mounts of sputum or yeasts in tissues;
central and eastern pleurisy; may be associated with exudates; organism forms branching septate microabscesses and
United States [Ohio verrucous or ulcerative skin lesions, hyphae with microconidia in culture at 30°C, pyogranulomas may
and Mississippi River subcutaneous nodules, osteolytic identification is usually confirmed by DNA be present in tissue
Valleys, Great Lakes] bone lesions, arthritis, prostatitis, hybridization; serologic tests have limited
and St. Lawrence river) and epididymitis utility
Coccidioides spp. (common Influenza-like syndrome or Endospores or spherules may be visible in Spherules with endospores
in the southwestern United pneumonia; also may cause calcofluor stains of wet mounts of sputum may be visible within
States) erythema nodosum or erythema or exudates; organism forms arthroconidia tissue; pyogenic and
multiforme, meningitis, and in culture at 30°C, identification is usually granulomatous (may be
disseminated disease confirmed by DNA hybridization; serologic caseous) responses can
tests are useful in both pulmonary and be found in tissue
disseminated diseases
Histoplasma capsulatum Influenza-like syndrome or Calcofluor or Giemsa stains may reveal Yeasts may be seen
(common in the Ohio and pneumonia; chronic progressive budding yeast or intracellular forms within intracellularly within
Mississippi River Valleys pulmonary infection in patients macrophages in respiratory specimens or macrophages and/or
in the United States, and with underlying lung disease; bone marrow; organism forms branching extracellularly as budding
parts of Central America) and disseminated disease in septate hyphae with tuberculate macroconidia forms; epithelioid
immunosuppressed patients in culture at 30°C, identification is usually granulomas may be
confirmed by DNA hybridization; serologic present
tests are useful in pulmonary disease; antigen
detection in serum and urine is particularly
useful in disseminated disease
Paracoccidioides brasiliensis Respiratory symptoms such as Calcofluor stains of wet preps of sputum or pus Multiple budding yeasts
(restricted to Central and productive cough and chest may reveal multiple budding yeast; organism detectable in tissues;
South America) pain; fever, weight loss, ulcerative forms branching septate hyphae in culture microabscesses and
granulomas of buccal, nasal, or at 30°C, identification usually confirmed by granulomas also may be
gastrointestinal mucosa may occur exoantigen tests or DNA-based assays present in tissue
Penicillium marneffei Disseminated disease in Grows as a septate mold at 30°C, often Yeast-like cells often contain
(restricted to Southeast immunosuppressed patients produces diffusible red pigment; identification cross-walls (divide by fission
Asia) confirmed by conversion to yeast form at 37°C rather than budding)
Filamentous fungi
Aspergillus spp. Aspergilloma (fungus ball) in Calcofluor white stains of respiratory or Septate hyphae with 45°
(A. fumigatus is most preexisting cavity; invasive biopsy material may reveal septate hyphae; branching are visible in
common pathogen) pulmonary aspergillosis with fever, culture isolates are usually identified by tissues (similar structures
dyspnea, and chest pain in patients colonial and microscopic morphology; the are seen in other invasive
with neutropenia and/or organ and serum galactomannan assay is useful for early fungal infections, as those
bone marrow transplantation; can detection of invasive pulmonary aspergillosis caused by Fusarium or
progress to disseminated disease Pseudoallescheria)
Mucor/Rhizopus Invasive pulmonary disease similar Calcofluor white stains of nasal or respiratory Broad, nonseptate hyphae
to aspergillosis; rhinocerebral specimens may reveal broad nonseptate with right-angle branching
mucormycosis begins with facial hyphae; organisms generally grow rapidly can be seen in tissue; often
pain and headache, can progress in culture but can be difficult to isolate from associated with necrosis
to invasion of the orbit and CNS tissue
Yeasts
Candida spp. Invasive mucosal infections such as KOH/calcofluor preps of mucosal scrapings Yeast, pseudohyphae,
esophagitis; disseminated disease in may reveal budding yeast, pseudohyphae, or or hyphae can be seen in
immunosuppressed or neutropenic hyphae; Candida grows well on routinely used infected tissue
patients agar and blood culture media
Cryptococcus neoformans Meningitis, pneumonia, skin lesions Antigen detection by latex agglutination Narrow-based budding
(in disseminated disease) of CSF is the most sensitive of the yeasts may be visible in
available tests; India ink smear may detect tissue (capsule stains with
yeasts; confirmatory culture using CSF is mucicarmine)
recommended
a
The dimorphic fungi can undergo reversible transition between mold forms and yeast forms. They grow as molds in the environment but replicate as yeast (or spherules)
in infected tissue.
Respiratory Virus Infections

Description
Many different viruses can cause upper and lower respiratory infections. Respiratory syncytial
virus (RSV) and influenza viruses cause large numbers of infections in the winter months. RSV is
the major cause of bronchiolitis in infants, and it can also cause serious infections in the elderly.
Influenza is often thought of as an infection of adults (in whom it causes a syndrome charac-
terized by rapid onset of fever, headache, and myalgias followed by upper respiratory symp-
toms). However, it also commonly infects children and may resemble RSV. Primary influenza
pneumonia is a rare but dangerous form of the infection. More commonly, the typical influenza
syndrome described above is followed several days later by a secondary bacterial pneumonia.
Parainfluenza viruses classically cause croup (tracheobronchitis), the clinical manifestations of
which often overlap those caused by RSV and influenza. Other important respiratory viruses
include adenoviruses, metapneumovirus, and coronaviruses, including the agent of SARS.
Because of the availability of antiviral agents that target influenza viruses, it has become impor-
tant to establish the specific cause of virus-like respiratory particularly in severely ill patients
and those with underlying cardiac and pulmonary diseases. Identification of the cause of severe
respiratory infections may also be needed for infection control activities. Immunosuppressed
patients are susceptible to all of the viruses described above. They are also at increased risk of
developing CMV pneumonitis.
Diagnosis The common respiratory

The common respiratory viruses can be identified by detecting specific viral antigens in naso- viruses can be
pharyngeal specimens (aspirates, washes, or swabs). Rapid tests for influenza can provide an identified by detecting
answer in less than 30 minutes, but their sensitivity is only 40% to 70% (specificity is >90%). specific viral antigens
in nasopharyngeal
Immunofluorescent assays in which a slide preparation is stained with labeled antibodies are
specimens (aspirates,
much more sensitive. However, they are more labor intensive and require a skilled observer. Iso-
washes, or swabs). Rapid
lation of respiratory viruses requires proper specimen collection to prevent the loss of viability
tests for influenza can
of the viruses before arrival in the laboratory. Traditional viral culture methods are sensitive, provide an answer in less
but respiratory viruses typically require 3 to 10 days in culture before they can be detected. Cen- than 30 minutes, but their
trifugation-enhanced culture, in which the organisms are centrifuged onto the cultured cells, sensitivity is only 40% to
can detect viruses in 1 to 2 days. Commercially available multiplexed nucleic acid amplification 70% (specificity is >90%).
assays (eg, RT-PCR) can detect several respiratory viruses in 1 to 8 hours and rival or exceed the
sensitivity of viral culture.
Laboratory methods for detecting respiratory viruses are summarized in Table 5–22.
INFECTIONS OF THE GASTROINTESTINAL TRACT

Overview
Although many organisms can cause infectious gastroenteritis, the clinical setting usually makes
it possible to focus on a small group of likely pathogens. This is particularly important since no 1
laboratory test can detect all of the potential agents, which include viruses, bacteria, and parasites.
Key factors to consider are whether the infection was acquired in the community or in a health
care facility, duration of symptoms, travel history, and whether the patient is immunosuppressed.
Community-acquired
Viruses Inducing Gastroenteritis diarrhea accompanied
by abdominal pain or
Description
systemic symptoms
In immunocompetent hosts, the majority of cases of community-acquired self-limited nausea, should be evaluated for a
vomiting, and/or diarrhea are caused by viruses (primarily rotaviruses, noroviruses, and enteric select group of bacterial
adenoviruses [types 40 and 41]). Laboratory testing is usually not performed unless there are pathogens, consisting of
large outbreaks that have epidemiologic significance, for example, outbreaks on cruise ships or Salmonella spp., Shigella
in health care facilities. Although enteroviruses are usually acquired by fecal–oral transmission, spp., Campylobacter spp.,
they generally cause systemic infections; gastroenteritis is not a prominent clinical manifestation. Shiga toxin-producing
CMV, especially in immunocompromised patients, can produce an explosive, watery diarrhea. E. coli, and Yersinia spp.
TABLE 5–28 Evaluation for Viral Infections of the Gastrointestinal Tract

Pathogen Clinical Findings Microbiology
Rotavirus Watery diarrhea, fever, vomiting (mostly in Direct antigen detection in stool specimens by immunoassay
infants and young children in winter) is the standard method
Adenovirus types 40/41 Watery diarrhea in infants; fever Direct antigen detection in stool specimens by immunoassay
(gastrointestinal adenoviruses are not culturable)
Cytomegalovirus (CMV) (in May have explosive diarrhea (can be watery CMV may be cultured from colonic biopsies; histopathology
immunosuppressed patients) or bloody); fever may reveal viral inclusions and inflammation (colitis)
Noroviruses Nonbloody diarrhea, vomiting, myalgias, RT-PCR is the method of choice for detecting noroviruses
and low-grade fever in clinical specimens
Diagnosis
Table 5–28 summarizes the clinical, radiologic, histopathologic, and laboratory findings associ-
ated with the gastrointestinal illnesses produced by rotavirus, adenovirus, CMV, and noroviruses.
Aerobic Bacterial Infections

Community-acquired diarrhea accompanied by abdominal pain or systemic symptoms should
be evaluated for a select group of bacterial pathogens, consisting of Salmonella spp., Shigella spp.,
Campylobacter spp., Shiga toxin-producing E. coli, and Yersinia spp. Recent travel or consumption
of raw shellfish would raise the possibility of Vibrio spp. All of these organisms are very unlikely to
be the cause of gastroenteritis in a patient who has been hospitalized for more than 3 days. Myco-
bacterial infections would need to be considered in profoundly immunosuppressed patients.
Table 5–29 describes some of the more common bacterial infections of the gastrointestinal tract.
Many of these organisms also can produce infections outside of the gastrointestinal tract.
C. difficile infection is Clostridium difficile Infections

frequently implicated Description
in antibiotic-associated
C. difficile infection is frequently implicated in antibiotic-associated diarrhea and is responsible
diarrhea. C. difficile
elaborates toxins A
for most cases of pseudomembranous colitis, a potentially life-threatening condition that requires
and B that induce fluid combined medical and surgical intervention. Antibiotics frequently implicated include ampicil-
secretion, mucosal lin, amoxicillin, cephalosporins, and clindamycin. In C. difficile colitis, there is a disruption of the
damage, and intestinal normal bacterial flora of the colon by the antibiotic treatment, after which there is colonization
inflammation and by C. difficile organisms. Colonization of the gastrointestinal tract occurs by the oral–fecal route.
produces heat-resistant C. difficile elaborates toxins A and B that induce fluid secretion, mucosal damage, and intestinal
spores that persist inflammation and produces heat-resistant spores that persist for months in the environment. The
for months in the organisms can be cultured from floors, toilets, bed pans, bedding, and all sites where patients with
environment. diarrhea from C. difficile infection have recently been treated.
C. difficile infection is typically acquired in the hospital by both infants and adults. Neonates
have an asymptomatic colonization, and most are resistant to the toxic effects of C. difficile infection.
Adults can also be asymptomatically colonized. Symptomatic C. difficile infection usually presents
with mild-to-moderate diarrhea and lower abdominal cramping. Symptoms often begin with anti-
biotic therapy, but may be delayed for several weeks after antibiotic therapy is initiated. Patients
who go on to develop pseudomembranous colitis experience more severe diarrhea, abdominal
tenderness, and systemic symptoms. Patients with advanced disease may present with a fulminant
life-threatening colitis that must be treated promptly to avoid perforation of the bowel wall.
Diagnosis
Since patients can be asymptomatically colonized with C. difficile, stool tests for toxigenic C. dif-
ficile should only be performed on patients with a compatible clinical presentation, such as those
with 3 or more loose stools in 24 hours. Table 5–30 summarizes the evaluation of patients for
C. difficile infection.
TABLE 5–29 Evaluation for Bacterial Infections of the Gastrointestinal Tract and for Peritonitis
Pathogen Clinical Findings Microbiology Additional Diagnostic Information
Bacterial infections
Campylobacter jejuni Acute enteritis with diarrhea (may Fecal wet mounts may reveal Leukocytes and erythrocytes often
be watery or bloody), fever, and darting motility of organisms; present in fecal smears; anti-GM1
abdominal pain; Guillain–Barre Gram-stained fecal smears have ganglioside antibodies may be
syndrome is an uncommon a sensitivity of 50%-75%; stool detected in post-Campylobacter
complication that may occur 2-3 cultures for C. jejuni must be Guillain–Barre syndrome
weeks following diarrhea incubated under microaerophilic
condition
Escherichia coli Watery or bloody diarrhea; hemolytic Routine stool cultures are useful For HUS strains, O and H serotyping
uremic syndrome (HUS) may be for suspected O157:H7 isolates; may be performed with cultured
associated with gastrointestinal special growth media may be isolates; the Shiga toxin can be
infection by Shiga toxin-producing required detected by immunoassay
isolates (eg, 0157: H7)
Mycobacterium avium- Watery diarrhea, abdominal pain, Blood cultures are the most likely Gastrointestinal symptoms precede
intracellulare (disseminated nausea, vomiting, weight loss, and to yield organisms; positive stool disseminated mycobacterial disease
infection in AIDS patients) night sweats culture by itself can represent in AIDS patients; lymph node, liver,
localized or disseminated or bone marrow biopsies may reveal
infection acid-fast organisms; bowel biopsies
not routinely performed
Salmonella enteritidis or Nonbloody diarrhea, fever, nausea, Routine stool cultures are useful; Serotyping of culture isolates is useful
typhimurium (salmonellosis) vomiting, and abdominal cramping blood cultures rarely positive (less for epidemiologic purposes; fecal
than 5%) smears usually have neutrophils
Salmonella typhi or paratyphi Fever, abdominal pain, Routine stool cultures are useful; Serologic tests are generally
(enteric or typhoid fever) hepatosplenomegaly, diarrhea, “rose blood cultures are 50%-70% not useful
spots,” weakness, and weight loss sensitive; bone marrow cultures
are 90% sensitive; duodenal fluid
collected by intestinal string can
be used for cultures
Shigella (shigellosis; bacillary Dysentery with abdominal pain Routine stool cultures used Direct fecal smears often contain
dysentery) and bloody diarrhea to detect organism abundant neutrophils; serologic
tests are not useful
Vibrio cholerae and other Mild or explosive watery diarrhea; Motile vibrios may be visible in Serotyping of organisms may
Vibrio spp. dehydration may be severe fresh fecal smears; stool cultures be performed
should include selective media
Yersinia enterocolitica Enterocolitis with diarrhea, Stool cultures on selective media Serologic tests for arthritis may
abdominal pain, and fever; reactive are necessary to permit growth of be useful for assessing patients
polyarthritis and erythema nodosum organisms for identification with polyarthritis
may occur after diarrhea
Clostridium difficile See Table 5–30

(antibiotic-associated colitis)
and Clostridium perfringens
(food poisoning)
Peritonitis
Primary peritonitis (usually Fever, abdominal pain, nausea, Gram stain and culture of Typically, peritoneal fluid protein
in children or patients with vomiting, and diarrhea peritoneal (ascitic) fluid is most is low (<3.5 g/L) and peritoneal
cirrhosis) likely to identify (in order of fluid leukocyte count is elevated
likelihood)—E. coli, Klebsiella (usually >1000/μL) with neutrophils
pneumoniae, Streptococcus >250/μL and pH <7.35
pneumoniae, enterococci
Secondary peritonitis (due Signs of sepsis with fever, Gram stain and culture of Peritoneal fluid studies less definitive
to perforation, appendicitis, tachycardia, tachypnea, and peritoneal fluid or aspirated in this setting; peripheral blood
cholecystitis) hypotension abscess material usually reveals leukocytosis often present; abdominal
mixed aerobic and anaerobic flora; ultrasound or CT scan may be useful
E. coli, Bacteroides fragilis, and for evaluation and identification of
Candida albicans commonly found suspected intra-abdominal abscesses
CT, computed tomography.
TABLE 5–30 Evaluation for Clostridial Infections of the Gastrointestinal Tract

Neutropenic Enterocolitis
Food Poisoning With Caused by Clostridium septicum
Diagnostic Test Clostridium difficile Colitis Clostridium perfringens, Type A and Other Organisms
Endoscopy Invasive and expensive; usually reserved for more Not useful Endoscopy is typically not
with biopsy severe cases performed, but if a sample
of suspicious of the bowel is removed, it
lesions will show the characteristic
inflammation and/or necrosis
Tests for toxins The presence of toxins A or B in diarrheal stools Various tests are available Not routinely performed
establishes the diagnosis of C. difficile colitis; the to detect the enterotoxin
tissue culture test for toxin B detects a cytopathic responsible for the toxic effect of
effect from filtrates of the diarrheal stool with C. perfringens in food poisoning;
sensitivities of ~80%; immunoassays for toxins A and these tests are performed in
B have relatively poor sensitivity (50%-60%); nucleic public health laboratories
acid amplification of the toxin genes provides rapid
detection as well as high sensitivity (~95%); tests for
C. difficile should only be performed on unformed
diarrheal stool, unless ileus is suspected
Stool cultures Most sensitive method; requires special media and Routine cultures of schools Not typically performed; because
must confirm that isolates produce toxin; patients samples are not useful; specialized bacteremia may be produced
may be asymptomatically colonized culture methods are performed in this illness, blood cultures for
in public health laboratories; 106 the clostridial organisms can be
spores per gram of stool or 105 collected
organisms per gram of suspected
food source supports the
diagnosis of food poisoning with
C. perfringens
Protozoal Infections
Description
Protozoa are a very diverse group of unicellular eukaryotic organisms that can be free-living or
parasitic. Many have 2 morphologic stages—trophozoites and cysts. Trophozoites, which are met-
abolically active feeding forms of the organism, may encyst within a protective coating to tolerate
harsh environments. The cyst is a dormant form of the protozoan, and can reemerge as a tropho-
zoite (for asexually reproducing organisms) when exposed to favorable conditions. For protozoa
that multiply by sexual reproduction, a zygote is formed from the fusion of 2 gametes. Encystation
of a zygote produces an oocyst that may contain 2 or more sporocysts, each with its own cyst wall.
Sporocysts contain sporozoites, infective forms of the organism.
The intestinal protozoa are divided into 5 main groups that differ in terms of epidemiology
and clinical presentation. Several of the groups contain pathogenic as well as nonpathogenic spe-
cies. Although the latter do not require treatment, their presence indicates that the individual has
been exposed to oral–fecal contamination.
• Flagellates: Pathogenic species include Giardia lamblia and Dientamoeba fragilis. Giardia
is the most common intestinal parasite in the United States. Sources of infection include
ingestion of contaminated water and person-to-person transmission in day care centers.
Giardia can cause diarrhea, abdominal cramps, bloating, and flatulence. Symptoms often
persist for more than 1 week.
The intestinal protozoa
• Amebas: Entamoeba histolytica is a major cause of intestinal infections worldwide,
are divided into 5 main particularly in tropical areas with limited sanitation facilities. Most infections in the
groups that differ in terms United States are acquired elsewhere. Clinical manifestations range from asymptomatic
of epidemiology and colonization to diarrhea, amebic colitis/dysentery, or extraintestinal abscess formation
clinical presentation. Most (usually in the liver). The diagnosis of amebiasis is complicated by the fact that E.
intestinal protozoa are histolytica is morphologically indistinguishable from the nonpathogenic E. dispar. Other
detected by examination nonpathogenic amebas include E. coli and Endolimax nana.
of stained stool • Coccidia: Human pathogens include Cryptosporidium spp., Cyclospora spp., and Isospora
specimens. belli. These organisms are members of the apicomplexans family and are related to the tissue
parasites T. gondii and Plasmodium spp. Cryptosporidium spp. are a relatively common cause
of infection in the United States. Outbreaks have been caused by contamination of drinking
water or recreational water such as pools or water parks. Cryptosporidium usually causes a
self-limited diarrhea in immunocompetent hosts, but it can cause severe persistent diarrhea
in AIDS patients. Cyclospora spp. have caused outbreaks linked to imported food, such as
raspberries. Isospora infections are usually only diagnosed in immunosuppressed patients.
• Ciliates: Balantidium coli is the only pathogenic ciliate.
• Microsporidia: Although these organisms are included in the section on protozoans,
recent phylogenetic analysis indicates that they are more closely related to the fungi.
Enterocytozoon bienusi causes self-limited diarrhea in normal hosts and chronic diarrhea
in AIDS patients in whom it can also spread to the biliary tract. Encephalitozoon spp. can
cause diarrhea and a variety of extraintestinal infections in immunosuppressed hosts.
Diagnosis
Most intestinal protozoa are detected by examination of stained stool specimens. Sensitive immu-
noassays are available for detecting antigens produced by Giardia and Cryptosporidium. Serology
can be useful for diagnosing invasive E. histolytica since E. dispar does not trigger an antibody
response. Table 5–31 describes infections produced by selected pathogenic protozoa. Protozoal
infections are commonly found within the gastrointestinal tract, but, as noted in the table, many
other organs and tissues can be infected.
Intestinal Helminth Infections

Description
Helminth (worm) infections in humans constitute a significant percentage of the global burden of
illness caused by infectious diseases. The helminths are multicellular organisms that are divided
into 3 groups: tapeworms (cestodes), roundworms (nematodes), and flukes (trematodes). Hel-
minths are typically enclosed by a protective coat, inside of which may be differentiated organ sys-
tems for digestion, neuromuscular control, and reproduction. Many helminths have complex life
cycles that involve 2 or more hosts. Helminths develop into adult worms and/or undergo sexual
reproduction in the definitive host, and they do not develop past the larval stage in intermediate
hosts. Infections in humans usually result from ingestion of eggs, penetration of intact skin by
infective larvae, or bites by insect vectors. Many helminth life cycles involve a stage in which the
larvae migrate through tissue. This migration can be relatively asymptomatic but can also have
serious clinical consequences depending on the type of helminth. These principles are illustrated
by the following 2 examples.
The definitive host for Echinococcus granulosis (a tapeworm) is the dog, which harbors the
adult tapeworm in its intestinal tract and excretes eggs in the feces. When an intermediate host,
such as sheep or humans, ingests these eggs, the eggs hatch in the intestinal tract and larvae pen-
etrate the intestinal wall and eventually form slowly expanding cysts in visceral organs such as
the liver or lungs.
Strongyloides stercoralis is 1 of the several nematodes that are acquired when infective larvae
present in warm moist soil penetrate human skin. The larvae migrate through tissue into the venous
circulation and are transported to the lungs where they invade the alveoli, are coughed up and swal-
lowed, and then develop into mature worms in the intestinal tract. The adult worms produce larva
that are shed in feces into the environment where they can complete their life cycle. Unlike other
nematodes, however, S. stercoralis larvae can also penetrate the gut wall or perianal skin and initi-
ate an autoinfective cycle that results in persistent low-level infection even after the host has left an
endemic area. If an infected patient subsequently receives immunosuppressive therapy, particularly
with corticosteroids, the patient can develop life-threatening Strongyloides hyperinfection syndrome
in which large numbers of nematode larvae migrate into the lungs and extraintestinal tissues.
Diagnosis
Infections with intestinal helminths are usually diagnosed by detection of eggs or larvae in
feces. For many of the helminths, the identification of the organism is based on the morphologic
characteristics of the organism and/or the eggs. These characteristics include size, shape, and
thickness of the egg wall, special structures such as knobs and spines, and the developmental stage
of the egg contents (eg, undeveloped, developing, or embryonated).
TABLE 5–31 Evaluation for Protozoal Infections

Histopathology/
Pathogen Clinical Findings Cytology Testing Comments
Microsporidia
Encephalitozoon Chronic, watery diarrhea; Spores are visible in Chromotrope-based staining D-Xylose and fat
and Enterocytozoon: dehydration, weight loss, fever, duodenal or biliary of stool specimens may be malabsorption are
microsporidiosis abdominal pain, and vomiting aspirates, or within used to detect spores common; serologic tests
enterocytes in small are not useful
intestinal biopsies;
electron microscopy
may be helpful
Amebas
Entamoeba histolytica Infection may be asymptomatic Cysts or trophozoites Cysts or trophozoites may be Serologic tests may be
or present as acute amebic or may be demonstrated visible in stool specimens used in detection of an
fulminant colitis with bloody in colonic scrapings or amebic liver abscess or
diarrhea; hepatic abscess can biopsies; if amebic liver intestinal amebiasis
be a late complication abscess is suspected,
abdominal imaging by
ultrasound or CT scan
should be performed
Naegleria fowleri Causes primary Brain biopsy not routinely Fresh CSF examination Purulent CSF with no
meningoencephalitis with recommended because (wet mount) for motile bacteria is common;
abrupt onset of headaches, CSF yields organisms, trophozoites; may be children or young adults
fever, nausea, vomiting, and even though brain biopsy Giemsa-stained; brain exposed to fresh water
pharyngitis; may be rapidly also may reveal organisms biopsies may be cultured are at risk
progressive
Acanthamoeba Causes keratitis with ocular Corneal biopsies may Giemsa, Gram, or Keratitis can be
pain and corneal ulceration; reveal organisms in calcofluor-stained smears subacute or chronic
also causes granulomatous patients with keratitis; of corneal scrapings may and is often associated
amebic encephalitis (GAE) brain or skin biopsy reveal amebas; culture of with soft contact
of nodules or ulcers organisms from corneal lens use
required for diagnosis scrapings or brain tissue
of GAE is possible
Ciliates
Balantidium coli Infection may be asymptomatic B. coli can invade the Wet preparation examination These organisms
or may cause severe diarrhea or colonic mucosa, with of fresh concentrated do not stain well,
dysentery; diarrhea may persist consequent ulcer stool will demonstrate making recognition
for weeks to months prior to formation; in such cases, the trophozoite and cyst and identification on
development of dysentery the organism is visible on forms; the organisms a permanent stained
histologic section are large and frequently smear difficult
can be seen under low
magnification
Flagellates
Giardia lamblia Acute or chronic watery Trophozoites may be Cysts or trophozoites may
diarrhea; nausea, anorexia, identified by endoscopic be visible in stool specimens;
low-grade fever, and chills brush cytology, mucosal direct antigen detection
smears, or histopathologic is also useful
examination of small
intestinal biopsy
Trichomonas vaginalis Vaginitis with excessive Visible on Pap smear Trichomonads may be Abundant neutrophils
discharge, dysuria, and observed in wet mounts are present in vaginal
dyspareunia of vaginal secretions wet mounts; nucleic
(60% sensitivity); acid hybridization or
endocervical or amplification tests
urethral cultures are are useful
most sensitive (exceed
90% sensitivity)

TABLE 5–31 Evaluation for Protozoal Infections (continued)

Histopathology/
Pathogen Clinical Findings Cytology Testing Comments
Coccidia
Cryplosporidium Watery, cholera-like diarrhea, Organisms may be visible Oocysts may be detected Serologic tests are not
parvum and C. hominis abdominal pain, nausea, fever, in small intestine biopsies, in concentrated specimens useful
and fatigue although many infections with acid-fast stain or DFA;
may be missed due to direct fecal antigen tests are
sampling variation useful
Cyclospora Watery diarrhea and Jejunal biopsy may Oocysts are visible in fresh Serologic tests are not
cayetanensis constipation, nausea, anorexia, show inflammation, stool; variable appearance available
abdominal cramping, and villous atrophy, or crypt with acid-fast stain of stool
weight loss hyperplasia; organisms specimen; oocysts show
may be detected with blue-green autofluorescence
acid-fast stain when excited at 365 nm
Isospora belli Profuse, watery diarrhea; Intestinal biopsies may Oocysts are visible in wet Serologic tests are not
abdominal pain, cramping, reveal organisms in smears of fresh or preserved available
weight loss, and low-grade sections stool; oocysts stain red with
fever; may be especially severe acid-fast stain
in HIV-infected patients
Toxoplasma gondii Lymphadenopathy or Tachyzoites often visible Tachyzoites often visible Serologic tests remain
mononucleosis-like syndrome in endomyocardial in CSF, amniotic fluid, or the standard for
in immunocompetent biopsies of heart bronchoalveolar lavage fluid; diagnosis to determine
adults; encephalitis, transplant recipients; antigens from the organism recent versus chronic
pneumonitis, or chorioretinitis lymph node pathology may be detectable in the infection; however,
in immunosuppressed is characteristic; brain serum; PCR may identify serologic studies
individuals; chorioretinitis biopsies lack sensitivity T. gondii DNA in respiratory lack sensitivity in
and/or neurologic findings in or amniotic specimens immunocompromised
congenital infections patients
Kinetoplastids (Not intestinal)
Leishmania: cutaneous Erythematous papules, Identification of Organisms from skin biopsy Serum antibody titers
and mucosal nodules, or ulcers; regional organisms in touch specimens may be cultured are not useful
leishmaniasis lymphadenopathy and fever preparations and sections in liquid media
of skin biopsy specimens
Leishmania: visceral Fever, malaise, weight loss, Fine needle aspiration Specimens obtained by fine High serum antibody
leishmaniasis hepatomegaly, splenomegaly of the spleen for touch needle aspiration of spleen, titers are present in
(kala-azar) preparation and culture is liver, and bone marrow may immunocompetent
>96% sensitive; organisms be cultured persons with visceral
may be visible in bone leishmaniasis
marrow aspirates
Trypanosomiasis, Chancre, intermittent fevers, Histopathologic Trypomastigotes visible in WBCs in CSF and an
African (sleeping lymphadenopathy, pruritic evaluation lacks peripheral blood smears, elevated CSF IgM titer
sickness caused by rash, and meningoencephalitis sensitivity chancre fluid, lymph node are useful for diagnosis
Trypanosoma brucei) or bone marrow aspirates of meningoencephalitis
Trypanosomiasis, Chronic illness highlighted Histopathologic In acute disease, parasites IgG serologic tests
American (Chagas by cardiac disease evaluation of heart may be detected in are used to diagnose
disease caused by (cardiomyopathy) and embolic or other tissues lacks peripheral blood or buffy chronic Chagas disease;
Trypanosoma cruzi) phenomena; lymphadenopathy sensitivity coat smears; lymph node, PCR-based detection
and chagoma occur in acute bone marrow aspirates, using peripheral
disease pericardial fluid, or CSF blood specimens is in
also may be examined development
CSF, cerebrospinal fluid; CT, computed tomography; PCR, polymerase chain reaction; DFA, direct fluorescent antibody.
Important information in the evaluation of a patient infected with helminths includes a his-
tory of possible exposure to the organism. Eosinophilia is commonly observed in patients with
helminth infections due to larval migration through tissues. The clinical findings, mode of trans-
mission to humans, aspects of microbiologic and serologic testing, and relevant radiologic find-
ings for selected helminth infections are presented in Table 5–32.
TABLE 5–32 Evaluation for Helminth Infections of the Gastrointestinal Tract

Mode of Transmission Microbiology and
Pathogen Clinical Findings to Humans Serology Testing
Tapeworms (Cestodes)
Diphyllobothrium latum (fish Diarrhea and abdominal pain; intestinal Ingestion of cysts in freshwater Characteristic operculate eggs
tapeworm) obstruction, vitamin B12 deficiency, and fish or proglottids (segments of the
pernicious anemia organism) may be present in
examination of feces
Echinococcus granulosus and Abdominal pain with hepatomegaly; Ingestion of eggs; due to Serologic tests available to assess
Echinococcus multilocularis confusion and headaches if CNS environmental contamination exposure
(restricted to high northern is involved; cysts in tissue such as from infected canines
latitudes) liver, lung, or brain; often discovered
incidentally on imaging
Taenia saginata (beef Abdominal discomfort, diarrhea, and Ingestion of the organisms Spherical eggs or gravid
tapeworm) intestinal obstruction in contaminated beef proglottids (segments of
the organism) often present
in feces
Taenia solium (pork tapeworm) Subcutaneous nodules, headaches, Ingestion of cysts in infected Proglottids (segments of the
generalized seizures if CNS is involved; pork leads to intestinal infection organism) may be visible in stool;
CNS cystic lesions with tissue (taeniasis) or ingestion of eggs serology useful in conjunction
displacement detected by CT or MRI (shed by human carrier) leads to with imaging for diagnosis of
invasive cysticercosis cysticercosis
Roundworms (nematodes)
Ascaris lumbricoides Loeffler syndrome (simple pulmonary Ingestion of embryonated Ovoid eggs usually present in
eosinophilia) in lungs with dyspnea, eggs; often hand-to-mouth examination of feces; developing
cough, and rales; eosinophilia in blood; transmission after contact with or adult worms may be present
intestinal obstruction or obstructive contaminated soil or surface in feces
jaundice
Enterobius vermicularis Usually children are infected; perianal Ingestion of eggs; often hand- Cellulose tape test performed in
(pinworm) and perineal pruritus to-mouth transmission after which tape adheres to eggs in
scratching perianal area or perianal folds; eggs transferred to
contact with contaminated slide for microscopy; anal swabs
surface; infection also may occur also useful
by inhalation of airborne eggs
in dust
Filariae (Wuchereria bancrofti Elephantiasis (lymphatic filariasis)— Injection of larvae—during Microfilariae may be visible in
for elephantiasis and lower extremity swelling and fevers; mosquito bite for W. bancrofti thick peripheral blood smears;
Onchocerca volvulus for onchocerciasis (river blindness)— and during black fly bite for skin snips or core biopsy of
onchocerciasis) cutaneous nodules and blindness O. volvulus nodules may reveal Onchocerca
microfilariae
Hookworms (Ancylostoma Intense pruritus, vesicular rash, Loeffler- Skin penetration by larvae, often Partially embryonated eggs
duodenale and Necator like syndrome, abdominal pain, bloody through the feet in contact with present in feces; larvae may be
americanus) diarrhea, and iron deficiency anemia contaminated soil present in feces
Strongyloides stercoralis Chronic infection—abnormal pain, Larvae penetration of skin First-stage larvae are often visible
intermittent urticarial rash, peripheral or colon; organism persists due in feces; larvae may be present in
blood eosinophilia; hyperinfection to autoinfective cycle sputum or duodenal aspirates
(immunosuppressed patient)—colitis,
abdominal distention, respiratory
distress, shock
Trichinella spiralis Gastroenteritis, fever, eosinophilia, Ingestion of larvae in Larvae may be detected
myositis, and circumorbital edema contaminated meat products in sediment from digested
following ingestion of raw pork or raw muscle tissue; antibodies to
bear meat (trichinosis) organism may be detected
with serologic tests
Trichuris trichiura (whipworm) Diarrhea, dysentery, and abdominal Ingestion of embryonated eggs Barrel-shaped eggs visible in feces
cramping; rectal prolapse may occur through hands, food, or drink
contaminated by contact with
infected soil or surfaces

TABLE 5–32 Evaluation for Helminth Infections of the Gastrointestinal Tract (continued)
Mode of Transmission Microbiology and
Pathogen Clinical Findings to Humans Serology Testing
Flukes (trematodes)
Fasciolopsis buski (intestinal Abdominal discomfort; travel Ingestion of metacercariae (larval Ellipsoidal eggs in feces or bile
fluke) or residence in Asia form of organism) in aquatic
plants
Liver flukes (Fasciola hepatica Hepatomegaly, cholangitis, hepatitis; Ingestion of metacercariae (larval Ellipsoid or ovoid eggs present
and Clonorchis sinensis) F. hepatica is worldwide; C. sinensis form of organism) in aquatic in feces
more common in Southeast Asian plants (for F. hepatica) and in
immigrants; with F. hepatica, hepatic freshwater fish (for C. sinensis)
nodules or linear tracks
Paragonimus westermani Cough with brownish sputum, Ingestion of metacercariae (larval Ovoid eggs are present in feces
(lung fluke) intermittent hemoptysis, pleuritic form of organism) in crayfish or and, less commonly, in sputum
chest pain, and eosinophilia freshwater crabs
Schistosoma haematobium Hematuria, granulomatous disease Penetration of intact human Eggs often visible in microscopic
(blood fluke) of the bladder, bladder carcinoma, skin by cercariae (larval form of examination of the urine
and secondary bacterial urinary tract organism), often during bathing,
infections; obstruction, hydronephrosis, swimming, or washing clothes in
or filling defects may be observed by contaminated water
renal ultrasonography or intravenous
pyelography
Schistosoma japonicum Nausea, vomiting, hemoptysis, Penetration of intact human Round to ovoid eggs in feces
(blood fluke) melena, hepatosplenomegaly, portal skin by cercariae (larval form of
hypertension, and esophageal varices organism) often during bathing,
swimming, or washing clothes in
contaminated water
Schistosoma mansoni Nausea, vomiting, hemoptysis, Penetration of intact human Lateral-spined eggs in feces that
(blood fluke) melena, hepatosplenomegaly, portal skin by cercariae (larval form of may be bloody or mucus-laden
hypertension, and esophageal varices organism) often during bathing,
swimming, or washing clothes in
contaminated water
CNS, central nervous system; CT, computed tomography; MRI, magnetic resonance imaging.
Food Poisoning Nausea and vomiting that

occurs 1 to 8 hours after
Nausea and vomiting that occurs 1 to 8 hours after eating can be caused by ingestion of bacterial
eating can be caused by
toxins that are already present in the food rather than by infection of the intestinal tract. The most ingestion of bacterial
common causes are enterotoxins produced by S. aureus (found in dairy and bakery products) toxins that are already
and Bacillus cereus (found in reheated fried rice). The condition is self-limited. C. perfringens present in the food rather
food poisoning results from the ingestion of food containing at least 108 enterotoxin-produc- than by infection of the
ing organisms. Often these are foods that have become grossly contaminated from storage over intestinal tract. The most
long periods at ambient temperature. This is particularly true of animal protein foods, such as common causes are
cooked meats and gravies. Most individuals experience watery diarrhea with abdominal cramps enterotoxins produced by
(B. cereus can cause a similar syndrome). Fatalities are rare, with spontaneous resolution of symp- S. aureus (found in dairy
toms within 6 to 24 hours. and bakery products) and
Bacillus cereus (found in
reheated fried rice).
Botulism
Description
Botulism is a neuroparalytic disease produced by potent toxins derived from Clostridium
botulinum. The toxins block the release of the neurotransmitter acetylcholine at peripheral cho-
linergic synapses. The most common cause of botulism in humans is ingestion of preformed
toxins in food contaminated with C. botulinum. Food products identified as sources of outbreaks
include home-canned vegetable products, fish products preserved by a variety of methods, and
sausage and ham preserved by salting rather than heating and then consumed without cooking.
Infant botulism, which affects children up to 35 weeks of age only, is a result of colonization of
TABLE 5–33 Laboratory Evaluation for Botulism

Laboratory Test Positive Result
Mouse bioassay This is the reference method for the detection of botulinum toxin; aliquots of serum,
feces, food extract, gastric fluid, or culture supernatant are injected intraperitoneally
into mice; control mice are injected with aliquots of the various samples containing
botulinum antitoxin; the mice are observed for the toxic effects of the botulism toxin;
if the mice receiving the botulinum antitoxin do not develop signs of botulism and
the mice not receiving the antitoxin do develop signs, the diagnosis is established
Bacterial culture The organism can be cultured anaerobically; the isolates must be shown to contain
toxins by the bioassay
Enzyme immunoassays Sensitive antigen detection assays are currently investigational
the intestinal tract by C. botulinum after the ingestion of viable spores. Wound botulism can occur
when C. botulinum contaminates deep wounds and secretes the toxin.
Patients suffering from botulism typically present with muscle weakness, difficulty in speak-
ing and swallowing, and blurred vision. Such patients can progress to symmetric descending
weakness and paralysis that can affect the diaphragm. Constipation, nausea and vomiting, and
abdominal cramping are also common presentations. Botulism toxin can be neutralized by anti-
sera raised against it, but the use of antitoxin may not reverse existing neuroparalysis. Recently
there has been concern about the potential use of botulinum toxin as a bioweapon.
Diagnosis
The laboratory confirmation of human botulism is established by detection of the toxin in the serum
or stool of an affected patient or in a sample of food consumed prior to onset of the illness. These
assays are only available in public health laboratories. Animal assays are still used because they can
detect very low levels of toxin. Isolation of C. botulinum from stools, gastric samples, or wound speci-
mens, in combination with the appropriate clinical signs and symptoms for botulism, also establishes
the diagnosis. For wound botulism, both serum and wound specimens should be tested for the pres-
ence of toxin and organisms. For infant botulism, stool samples are required for analysis.
The laboratory diagnosis is described in Table 5–33.
PYELONEPHRITIS AND URINARY TRACT INFECTIONS

Description
UTIs can be divided into 3 categories:
• Uncomplicated infections of the lower urinary tract involving the bladder and/or urethra
• Uncomplicated infections of the upper urinary tract, or pyelonephritis, involving the
ureters, renal pelvis, and kidney
• Complicated UTIs involving various sites within the urinary tract
Acute symptomatic Acute symptomatic uncomplicated lower UTIs are very common in women. The typical
uncomplicated lower symptoms are painful urination (dysuria), urgency, and frequency. Approximately 80% of these
UTIs are very common infections are caused by E. coli. Uropathogenic E. coli are genetically distinct from other intestinal
in women. The typical strains and possess virulence factors that facilitate colonization of the urinary tract epithelium.
symptoms are painful Other enteric gram-negative rods, such as Proteus spp. and Klebsiella spp., and gram-positive
urination (dysuria), cocci including Staphylococcus saprophyticus and enterococci can cause uncomplicated UTIs. The
urgency, and frequency. high incidence of UTIs in women is probably due to the relatively short length of the urethra and
Approximately 80% its proximity to the anus and the genital tract. Risk factors for uncomplicated lower UTI in young,
of these infections are sexually active women include intercourse and diaphragm and spermicide use. UTIs are uncom-
caused by E. coli.
mon in men until after the age of 50 years. UTIs in children are also more common in females.
They can be associated with constipation, incomplete or infrequent voiding, sexual abuse, and
anatomic defects within the urinary tract.
Acute uncomplicated pyelonephritis (upper UTI) is usually due to an ascending infection
that begins in the bladder. The main clinical features are fever and chills, nausea, vomiting, and
abdominal pain. Costovertebral angle tenderness is usually present. Risk factors for pyelone-
phritis include the presence of renal stones, obstruction of urine outflow, vesicoureteral reflux,
pregnancy, anatomic abnormalities of the kidney and urinary tract, and urinary catheteriza-
tion. Intrarenal infections are often the result of hematogenous dissemination of S. aureus or
Candida spp.
Complicated UTI refers to infections in patients with a variety of underlying conditions such The laboratory diagnosis
as anatomic or functional urologic abnormalities, stones, or obstruction. Imaging studies are of a UTI involves tests
often useful for identifying the underlying problem. Other predisposing factors are indwelling to detect WBCs in the
catheters or urologic instrumentation, immunosuppression, renal disease, and diabetes. These urine (pyuria) and tests
infections are typically caused by hospital-acquired bacteria including Klebsiella spp., Proteus to detect bacteria in the
spp., Morganella morganii, P. aeruginosa, enterococci, staphylococci, and yeast. urine (bacteriuria).
Asymptomatic bacteriuria occurs in 3% of women; of these, 10% will develop UTIs. A higher
incidence of UTIs is found in the elderly population where 10% to 15% of women older than 60
years suffer from recurrent UTIs.
Diagnosis
The laboratory diagnosis of a UTI involves tests to detect WBCs in the urine (pyuria) and tests to
detect bacteria in the urine (bacteriuria).
Rapid detection of WBCs and bacteria in the urine can be performed using a urine dipstick.
The WBCs are detected with a dipstick pad containing leukocyte esterase reagents and some bac-
teria can be detected by their ability to convert nitrate to nitrite (see Chapter 18 for a discussion
of urinalysis). WBCs and bacteria also can be identified and counted in a microscopic analysis of
the urine. A urine WBC count of >5 leukocytes per high-power field is defined by most authors
as significant pyuria. The level of bacteriuria in the 3 categories of UTIs varies.
Urine culture is the gold standard for the diagnosis of UTIs, although it may not be nec-
essary for uncomplicated outpatient UTIs. Unlike most other specimen types, urine is always
cultured using a quantitative procedure because interpretation of the results depends on both
the type and the number of organisms in the specimen. Because urine passes through the distal
urethra, which is colonized with a variety of gram-negative rods and other organisms, isolation of
bacteria from a midstream clean catch urine specimen does not automatically establish the pres-
ence of infection. Significant bacteriuria is often defined as the presence of ≥105 colony-forming
units (CFU)/mL; however, many patients with a urethral syndrome can have lower counts. The
presence of 3 or more organisms with none predominating indicates contamination, and a new
specimen should be collected. Rapid specimen transport and refrigeration of stored specimens is
important because bacteria can replicate in urine that is left at room temperature, unless a preser-
vative is used, leading to overgrowth of contaminants and inaccurate colony counts. A summary
of the laboratory evaluation for UTI is presented in Table 5–34.
TABLE 5–34 Evaluation for Urinary Tract Infection (UTI)

Laboratory Test/ Uncomplicated Lower Complicated Urinary
Clinical Feature Urinary Tract Infection Uncomplicated Pyelonephritis Tract Infection
Site of infection Bladder and urethra Ureters, renal pelvis, and kidney Varies
Risk factors Intercourse and diaphragm/spermicide Include risk factors for both uncomplicated Structural or functional abnormalities
use lower UTI and complicated UTI in the urinary tract
Symptoms Frequency, urgency, and dysuria Frequency, urgency, dysuria, flank pain, Depend on the site of infection
and fever
Level of pyuria >5 WBC per high-power field using >5 WBC per high-power field using a fresh >5 WBC per high-power field using
a fresh urine specimen or a positive urine specimen or a positive leukocyte a fresh urine specimen or a positive
leukocyte esterase dipstick test result esterase dipstick test result leukocyte esterase dipstick test result
Level of For women without symptoms, >104 CFU/mL ≥105 CFU/mL

bacteriuria (CFU) >105 CFU/mL on 2 consecutive specimens;
for women with symptoms, >102 CFU/mL;
for men with symptoms, >103 CFU/mL
CFU, colony-forming unit.
TABLE 5–35 Infections of the Male Genital Tract

Site of Infection Common Causative Organisms
Seminal vesicles Gram-negative bacilli and Neisseria gonorrhoeae
Epididymis Chlamydia, gram-negative bacilli, N. gonorrhoeae, Mycobacterium tuberculosis
Prostate gland Gram-negative bacilli, enterococci (and staphylococci), and N. gonorrhoeae
INFECTIONS OF THE MALE GENITAL TRACT

Epididymitis is most often caused by a sexually transmitted disease such as gonorrhea or Chla-
mydia infection (discussed in a later section). However, it can also be caused by enteric gram-
negative rods or Pseudomonas in patients with underlying urinary tract disease.
Acute bacterial prostatitis presents with urinary tract symptoms, a tender prostate, and is
often accompanied by systemic findings such as fever. Chronic infection of the prostate due to
gram-negative rods or gram-positive cocci is often asymptomatic, but it can serve as a source of
recurrent symptomatic bacteriuria. Chronic pelvic pain syndromes have also been attributed to
The most common chronic prostatitis, but often the etiology is unclear. Granulomatous prostatitis caused by extra-
infection of the testicle pulmonary TB or systemic fungal infections produces nodular lesions that can mimic prostatic
is viral orchitis that is carcinoma. Histologic examination of a biopsy specimen would distinguish these possibilities.
usually caused by mumps Table 5–35 provides an association between site of infection in the male genital tract and com-
or coxsackieviruses. mon causative organisms.
The most common infection of the testicle is viral orchitis that is usually caused by mumps or
coxsackieviruses. Mumps is an acute viral disease that causes painful enlargement of the salivary
glands, particularly the parotid glands. The virus is transmitted via respiratory droplets. Orchi-
tis in postpubertal males is often due to mumps, although the incidence is low due to vaccina-
tion. Laboratory diagnosis is important for epidemiologic investigations. The laboratory tests for
mumps are summarized in Table 5–36.
INFECTIONS OF THE FEMALE GENITAL TRACT

Infections of the female genital tract include vaginitis, vaginosis, and cervicitis/pelvic inflamma-
tory disease (infection of the uterus, fallopian tubes, and adjacent structures). As with infections
of the male genital tract, many of these infections are due to sexually transmitted diseases that are
discussed in a later section.
The primary symptom of vaginitis is pruritus that can be accompanied by a discharge. The
most common cause is the yeast Candida albicans. Trichomonas vaginalis (a protozoan) can cause
a similar syndrome. The chief complaint in bacterial vaginosis is vaginal odor. In the past this
TABLE 5–36 Laboratory Evaluation for Mumps

Serology A positive mumps IgM assay is useful for diagnosis of acute mumps infection.
Measurement of a mumps IgG seroconversion (from negative to positive) in acute and
convalescent specimens or a 4-fold rise in mumps IgG titer also supports the diagnosis
PCR RT-PCR is useful for detecting viral RNA in oropharyngeal secretions (eg, parotid duct
fluid), urine, and CSF
Culture Mumps virus grows slowly; it can be isolated from oropharyngeal secretions, urine,
and CSF. Typing of viral isolates is important for epidemiologic studies
TABLE 5–37 Infections of the Female Genital Tract

Common Etiologic
Disease/Condition Agent(s) Clinical Features Laboratory Diagnosis
Vulvovaginitis Candida albicans, Pruritus, irritation, external Microscopy after treating specimen with 10% KOH to
Trichomonas vaginalis dysuria, vaginal discharge reveal yeast and hyphal forms; wet mount to detect
(especially with T. vaginalis) motile trichomonads; culture; nucleic acid detection
Vaginosis Polymicrobial (multiple Vaginal odor, vaginal discharge Vaginal discharge pH >4.5; “fishy” odor after addition of
anaerobes and 10% KOH; “clue cells” (vaginal epithelial cells coated with
Gardnerella vaginalis) coccobacilli) on wet mount; or Gram stain with decreased
gram-positive rods and increased gram-negative or
variable coccobacilli
Cervicitis, pelvic Chlamydia trachomatis, Cervicitis is often Culture or nucleic acid amplification of N. gonorrhoeae
inflammatory disease Neisseria gonorrhoeae asymptomatic; PID is associated and C. trachomatis from cervical swab; nucleic acid
(PID) with lower abdominal pain, amplification of urine also useful but less sensitive
vaginal discharge, dysuria,
and dyspareunia; long-term
sequelae can include infertility
and ectopic pregnancy
condition was ascribed to overgrowth of Gardnerella vaginalis, but the current view is that it The primary symptom of
results from a disruption of the normal vaginal flora in which lactobacilli (gram-positive rods) are vaginitis is pruritus that
largely replaced by a mixture of gram-negative coccobacilli. can be accompanied by
Table 5–37 briefly describes the etiologic agents, clinical features, and laboratory diagnosis of a discharge. The most
vaginitis, vaginosis, and pelvic inflammatory disease. Genital herpes and other sexually transmit- common cause is the
ted diseases are discussed in a subsequent section. yeast Candida albicans.
Organisms that infect or colonize the female genital tract can also cause infections of Trichomonas vaginalis
the newborn. Beta-hemolytic streptococci belonging to Lancefield group B (GBS) are also (a protozoan) can cause
a similar syndrome.
known as S. agalactiae. These organisms often asymptomatically colonize the gastrointesti-
nal and female genital tracts; however, they are also an important cause of neonatal sepsis
and meningitis (E. coli capular type K1 is another common cause of this type of infection).
Risk factors associated with early onset neonatal infection include maternal colonization with
GBS, premature rupture of membranes, chorioamnionitis, and previous delivery of an infected
infant. Pregnant women are routinely screened during the third trimester at 35 to 37 weeks
for colonization with GBS. This can be done by culture of vaginal and rectal swabs or nucleic
acid amplification. Women who are colonized (or have the risk factors listed above) are given
antibiotics during delivery to prevent neonatal infections. Chlamydia trachomatis, Neisseria
gonorrhoeae, and Herpes simplex can also be transmitted to the newborn during delivery and
cause serious infections.
SEXUALLY TRANSMITTED DISEASES

Syphilis
Description
Syphilis is a multisystem infectious disease that has prominent dermatologic and neurologic
manifestations. It is caused by Treponema pallidum, a thin elongated bacterium known as a spiro-
chete. T. pallidum is typically spread through contact with infectious lesions during sexual activ-
ity. Transmission occurs in about one third of patients exposed to early syphilis. Primary skin
lesions, also known as chancres, usually develop within 3 weeks after initial exposure. Primary
syphilis is the stage defined by the presence of lesions at the site of inoculation. Secondary syphilis
is the stage of hematogenous dissemination of T. pallidum, with widespread physical findings and
constitutional signs and symptoms. The signs and symptoms include rash, alopecia, condylomata
lata, and shallow painless ulcerations of mucous membranes known as “mucous patches.” Even in
the absence of treatment, the signs of primary and secondary syphilis spontaneously resolve, and
patients enter a latent stage of infection. Manifestations of tertiary syphilis develop in approxi-
mately 30% of untreated patients after a variable period of latency. The manifestations of tertiary
syphilis involve cardiovascular and/or neurologic and ophthalmic abnormalities. Neurologic
involvement, however, is not limited to patients in the tertiary stage of the disease. The clinical
manifestations of neurosyphilis include meningitis, general paresis, and tabes dorsalis. Congeni-
tal syphilis can occur in newborns whose mothers have syphilis.
The number of cases of primary and secondary syphilis in the United States was relatively
stable from the early 1960s to the mid-1980s with 20,000 to 30,000 cases per year. With the
appearance of AIDS and the decline of public health programs, the number of cases of primary
and secondary syphilis in the United States increased to more than 50,000 by 1990; however,
by 2000 it had declined by 80%. More recently there has been a gradual increase in the number
of cases.
Diagnosis
T. pallidum cannot be T. pallidum organisms are too narrow to be visualized by standard light microscopy, but they can
cultured on microbiologic be seen by dark-field microscopy. This technique requires considerable expertise to distinguish
media. As a result, T. pallidum from nonpathogenic treponemes and other artifacts, and currently it is rarely available.
serologic testing is T. pallidum cannot be cultured on microbiologic media. As a result, serologic testing is the
the most widely used most widely used approach for the diagnosis of syphilis. Two types of tests are routinely used.
approach for the Nontreponemal screening tests for syphilis include the Venereal Disease Research Laboratory
diagnosis of syphilis. (VDRL) and rapid plasma reagin (RPR) tests. These assays detect antibodies that react with an
Nontreponemal screening antigen composed of cardiolipin and other lipids. A single reactive test requires supplemental
tests for syphilis include historical, clinical, or laboratory information to provide a diagnosis of syphilis, as there are many
the Venereal Disease
biologic causes of a false-positive VDRL or RPR.
Research Laboratory
Positive screening test results are routinely confirmed with more specific tests that detect
(VDRL) and rapid plasma
reagin (RPR) tests. Positive
antibodies that react with T. pallidum antigens (ie, treponemal tests). The fluorescent trepone-
screening test results are mal antibody absorption test (FTA-ABS) uses indirect immunofluorescence to detect the binding
routinely confirmed with of the patient’s antibodies to T. pallidum organisms fixed onto a microscopic slide (the patient’s
more specific tests that serum is first preabsorbed with a nonpathogenic treponeme to increase the specificity of the test).
detect antibodies that The microhemagglutination assay for T. pallidum test (MHA-TP) measures the ability of serum
react with T. pallidum antibodies to agglutinate RBCs that are coated with formalin-fixed T. pallidum. Because these
antigens. assays are more expensive and/or technically demanding than the screening tests, they have tra-
ditionally been used to confirm a positive nontreponemal test rather than being used for initial
evaluation. The introduction of high-throughput EIAs utilizing T. pallidum antigens has led to a
reevaluation of the standard testing algorithm.
The treponemal tests are specific and sensitive, but they do not distinguish current infection
from past infection. Although the nontreponemal tests are less specific, they are still very useful
because changes in the antibody titer are used to monitor the response to therapy.
Diagnosis of syphilis in newborns is complicated by the fact that they can have substantial
quantities of antitreponemal IgG as a result of transfer of this IgG from the maternal circula-
tion to the fetus. A serologic diagnosis of congenital syphilis in the neonate can be confirmed if
antitreponemal IgM, made by the fetus, is found in the neonatal circulation. An infant RPR titer
greater than the maternal RPR titer also supports a diagnosis of congenital syphilis.
The laboratory tests used in the diagnosis of primary, secondary, latent and tertiary, and con-
genital syphilis are shown in Table 5–38.
Gonorrhea
Description
Gonorrhea, an infection with the organism N. gonorrhoeae, is a major cause of morbidity as a
sexually transmitted disease, primarily because of complications of the initial infection. These
complications include ascending pelvic infections in women, epididymo-orchitis in men, and
disseminated gonococcal infections in women and men. Infants born to untreated mothers can
also develop ophthalmia neonatorum. The clinical symptoms of gonorrhea include dysuria, ure-
thral and/or vaginal discharge, and pelvic pain. Gonorrhea is generally more symptomatic in
men than in women. Clinical features include urethral discharge and mucopurulent cervicitis,
TABLE 5–38 Evaluation of Syphilis

Laboratory Test Primary Syphilis Secondary Syphilis Latent and Tertiary Syphilis Congenital Syphilis
Dark-field microscopy In early primary stage, If exudative secondary stage Exudative lesions are absent, Exudative lesions are
(from wet prep of when other tests are lesions are present, this test so this test cannot be absent, so this test
exudate obtained less sensitive, this test is useful performed cannot be performed
directly from chancre is useful
or lesion)
Rapid plasma reagin Lag in nonspecific Rapid, inexpensive screening Screening test for both latent Maternal IgG antibodies
(RPR) test serologic response test; incidence of biologic and tertiary stages; VDRL cross placenta
results in markedly false-positivity ranges from recommended over RPR and complicate
reduced sensitivity 0.3% to 1%; positive results to diagnose neurosyphilis interpretation; need
in primary syphilis must be confirmed by other when using a CSF specimen to compare infant and
antitreponemal serology tests; maternal titers
useful for treatment follow-up
by assessing titer of antibody
Venereal Disease Lag in nonspecific Rapid, inexpensive screening Screening test for both latent Maternal IgG antibodies
Research Laboratory serologic response test; incidence of biologic and tertiary stages; positive cross placenta
(VDRL) test results in markedly false-positivity ranges from CSF VDRL (sensitivity 30%- and complicate
reduced sensitivity 0.3% to 1%; positive results 70%) is sufficient to diagnose interpretation; need
in primary syphilis must be confirmed by neurosyphilis, but negative to compare infant
antitreponemal serologies; CSF VDRL does not exclude and maternal titers
useful for treatment follow-up diagnosis
by assessing titer of antibody
Fluorescent treponemal Used as a confirmatory Useful as a confirmatory Useful as a confirmatory There is a useful
antibody test with diagnostic test or in diagnostic test in RPR- or diagnostic test in RPR- or modification of
absorptions (FTA-ABS) lieu of RPR or VORL; VDRL-positive patients; overall VDRL-positive patients; the standard test
sensitivity of 80%-85% in sensitivity is approximately 98%; overall sensitivity is that detects only
primary syphilis specificity >98% (but does not approximately 98%, but it is neonatal or infant
distinguish syphilis from yaws or reduced in late latent phase IgM antitreponemal
pinta); not useful for monitoring antibody, known as
therapy the IgM FTA-ABS assay
Microhemagglutination Useful as confirmatory Useful as confirmatory Useful as confirmatory Maternal

assay for Treponema diagnostic test or in diagnostic test in RPR- or diagnostic test in RPR- or antitreponemal IgG
pallidum (MHA-TP) lieu of RPR or VDRL; VDRL-positive patients; high VDRL-positive patients; antibodies cross
sensitivity of 80%-85% specificity (but does not sensitivity reduced in late placenta, rendering
in primary syphilis distinguish syphilis from latent phase test ineffective
yaws or pinta); not useful
for monitoring therapy
Enzyme immunoassay Useful as confirmatory Not useful because IgM level Not useful because serum This test is useful for
for specific detection of diagnostic test, diminishes several weeks after IgM levels are negligible in making the diagnosis,
anti-T. pallidum IgM especially in untreated infection latent and chronic infection because, unlike IgG,
primary syphilis maternal IgM does not
cross the placenta
Enzyme immunoassay Useful as confirmatory Useful as confirmatory Useful as confirmatory Maternal

for specific detection of diagnostic test, diagnostic test, with sensitivity diagnostic test, with antitreponemal
anti-T. pallidum IgG confirmatory, especially and specificity approaching sensitivity and specificity IgG antibodies
in untreated primary 100%; can also be used in approaching 100%; can cross placenta
syphilis “reverse algorithm”a also be used in “reverse and complicate
algorithm”a interpretation
Western blot to detect Useful as confirmatory Not useful because IgM level Not useful because serum This test is useful in
anti-T. pallidum IgM diagnostic test especially fades several weeks after IgM levels are negligible in diagnosis, as maternal
in untreated primary infection latent and chronic infection IgM does not cross the
syphilis; IgM becomes placenta
detectable 2 weeks after
first chancre appears
Western blot to detect May be useful as a May be useful as a confirmatory May be useful as a Maternal
total anti-T. pallidum IgG confirmatory diagnostic diagnostic test in RPR- or confirmatory diagnostic antitreponemal IgG
test, although FTA-ABS VDRL-positive patients; overall test in RPR- or VDRL-positive antibodies cross
and MHA-TP are simpler sensitivity of approximately 90% patients placenta, rendering test
and more rapid and specificity of 100% ineffective
a
Data from Loeffelholz MJ, Binnicker MJ. It is time to use treponema-specific antibody screening tests for diagnosis of syphilis. J Clin Microbiol. 2012;50(1):2–6.
TABLE 5–39 Sample Collection Site by Patient Type for Neisseria gonorrhoeae
Specimen Patient
a
Urine Symptomatic (or at-risk) male or female
Urethral exudate Symptomatic male
Urethral swab if no exudate can be expressed Symptomatic male
Anorectal and pharyngeal swab Male or female with rectal or pharyngeal exposure
Conjunctival swab Infant with conjunctivitis
Blood and synovial fluid Male or female patient presenting with arthritis and/or
dermatitis and suspected of prior gonococcal infection
Swab from endocervical canal Female suspected of infection

a
For nucleic acid amplification, not culture.
respectively. Untreated asymptomatic individuals serve as a reservoir for N. gonorrhoeae. Trans-

mission from males to females is more efficient than in the reverse direction. Pharyngeal infec-
tions of N. gonorrhoeae are typically asymptomatic.
The incidence of gonorrhea in the United States peaked in 1978 and declined approximately
75% through the late 1990s. Since then it has leveled off. Most cases are reported in men because
they are more symptomatic than women. Individuals with gonorrhea have a high rate of other
sexually transmitted diseases and therefore require complete screening. Ascending pelvic infec-
tions that occur in 10% to 20% of acutely infected women can result in infertility and ectopic
pregnancy.
N. gonorrhoeae requires Diagnosis

a nutrient-rich selective
The gold standard for diagnosis continues to be growth of the organism in culture. N. gonorrhoeae
agar for successful
requires a nutrient-rich selective agar for successful culture and an incubation period of up to
culture and an incubation
period of up to 48 hours
48 hours for colony formation. Due to the fastidious nature of the organism, false-negative results
for colony formation. frequently occur as a result of poor specimen handling and delayed transport. As a result of these
Due to the fastidious limitations, nucleic acid amplification is now widely used to diagnose gonorrhea. These NAAT
nature of the organism, assays are as sensitive as culture when performed on cervical or male urethral swabs and provide
false-negative results rapid results. Several of the assays are also approved for use on urine, although the sensitivity is
frequently occur as a less for this type of specimen. These assays have high specificity, but caution must be used when
result of poor specimen interpreting positive results in a low-prevalence population. Culture is still recommended for
handling and delayed nongenital sites. The samples collected for analysis depend on the site most likely to be infected
transport. and the sex of the patient (Tables 5–39 and 5–40).
Chlamydial Infections
Description
Chlamydiae are gram-negative, nonmotile, obligate intracellular bacteria. C. trachomatis is the
most common cause of sexually transmitted disease in North America. It is also the agent of
trachoma, a major cause of preventable blindness worldwide. Chlamydophila pneumoniae causes
a respiratory infection that is similar to Mycoplasma infection. C. psittaci, which is common in
certain birds and can be spread to humans via aerosolized feces, causes psittacosis, a respiratory
and/or systemic infection.
C. trachomatis produces up to 4,000,000 infections each year in the United States as a
sexually transmitted disease. Groups at increased risk for C. trachomatis infection include men
or women who have had a new sexual partner or more than 1 sexual partner in the last year
and sexually active women using barrier contraceptive methods. Approximately one third of
infected males and half of infected females may have asymptomatic or mild infections. Sub-
clinical infection and scarring of the fallopian tubes with subsequent infertility is 1 of the major
complications of Chlamydia infections. C. trachomatis can also infect newborns during delivery
TABLE 5–40 Evaluation for Neisseria gonorrhoeae Infection

Gram stain and culture Gram-negative kidney-bean-shaped diplococci (within neutrophils and
extracellular) on Gram stain; the sensitivity of Gram stain smears for detection
of gonorrhea varies from 40% to 95% depending on the patient and the site
of collection
Culture of the organism The gold standard method, usually requires 1-2 days to become positive; it is
not 100% sensitive, especially when there are delays in specimen collection and
transport
DNA probe hybridization These tests provide rapid turnaround time because there is no need to grow
tests the organism in culture; a disadvantage is the lack of an isolate for subsequent
susceptibility testing; these tests have largely been supplanted by amplification
that is more sensitive
DNA amplification tests These tests provide direct detection of N. gonorrhoeae and/or C. trachomatis in
clinical specimens using polymerase chain reaction (PCR), strand displacement
amplification (SDA), or transcription-mediated amplification (TMA); these tests
provide rapid results but are costly; their major advantages are that they can be
used with urine specimens and swab specimens, are the most sensitive assays
available, and have a high specificity (≥98%)
and cause conjunctivitis and pneumonia. Lymphogranuloma venereum (LGV), a disease char-
acterized by tender inguinal lymphadenopathy and often proctitis, is caused by specific serovars
of C. trachomatis.
Diagnosis
Direct detection of chlamydial DNA using NAAT assays is now the preferred method for diag-
nosing genital C. trachomatis infections due to the high sensitivity and specificity of these assays.
These tests use PCR, strand displacement amplification (SDA), or transcription-mediated ampli-
fication (TMA) to amplify C. trachomatis genes. They have largely replaced other nonculture
methods such as antigen detection.
Diagnosis of a chlamydial infection also can be made on the basis of a positive culture of
the organism from infected sites. This requires cells in which the organism proliferates and is a
labor-intensive procedure. Careful sample collection and specimen transport are important in the
maintenance of viable organisms for culture. Although culture is less sensitive than NAAT assays,
it is still used in medicolegal situations. Commercial NAAT assays may be positive in patients
with LGV, but they do not distinguish specific LGV serovars nor are they currently FDA-approved
for rectal specimens. Serologic tests for anti-Chlamydia antibodies involving complement fixation
or immunofluorescence detect antichlamydial IgG or IgM in the serum and are sometimes used
to support the diagnosis of LGV.
Table 5–41 summarizes the tests available to diagnose Chlamydia infections.
Herpes Simplex Virus Infections

Description
HSV is a double-stranded DNA virus surrounded by a lipid envelope and is usually transmitted
by person-to-person contact. The virus initially causes a productive infection of epithelial cells
and then establishes a latent infection in sensory ganglia for the lifetime of the host. It can later
reactivate and produce active infections. The classic pattern of infection is a group of recurring
vesicles on an erythematous base; however, HSV infection is often asymptomatic, and lesions
occur in a minority of infected patients. Individuals infected with HSV are potentially contagious,
whether or not lesions are visible. HSVs can be subdivided into HSV type 1 and type 2. Oral her-
pes infections, which are typically present as cold sores, are primarily caused by HSV-1. Genital
herpes infections are primarily caused by HSV-2. It is estimated that 50,000,000 individuals in the
United States have genital HSV infection. Transmission of genital herpes occurs during sexual
TABLE 5–41 Evaluation of the Patient for Chlamydial Infection

Laboratory Test Chlamydia trachomatis Chlamydia psittaci Chlamydia pneumoniae
Culture of the organism Organisms commonly require 48-72 Organisms commonly require Organisms are difficult to grow in
hours to grow in cultured cells; the 5-10 days to grow in cultured cultured cells; in positive cultures,
intracytoplasmic inclusions are best cells; the intracytoplasmic the intracytoplasmic inclusions are
visualized with fluorescein-conjugated inclusions are best visualized with best visualized with fluorescent
monoclonal antibodies fluorescent antibodies antibodies
Microscopic examination Useful in the diagnosis of acute Not useful Not useful
of stained smear from neonatal inclusion conjunctivitis
potentially infected site (sensitivity >90%)
Direct immunofluorescence Test performed in minutes, but the Not specific for C. psittaci Not specific for C. pneumoniae
(DIF) of sample from result is dependent on the skill of
potentially infected site the person performing the assay;
most useful for cervical and urethral
specimens
Enzyme-linked Less sensitive and less specific in Cross-reactions with normal Cross-reactions with normal
immunoassay (EIA) using cervical infections than the DIF test respiratory flora limit its utility respiratory flora limit its utility
sample from potentially for the detection of C. trachomatis,
infected site although it can be modified by using
different antibodies to improve
sensitivity and specificity
Serologic test using Not useful for the detection of Useful in the diagnosis of Useful in the diagnosis of primary
complement fixation (CF) trachoma, neonatal infections, psittacosis if there is a 4-fold infection if there is a 4-fold increase
technique inclusion conjunctivitis, and genital increase in titers between acute in titers between acute and
infections and convalescent serum samples convalescent serum samples
Serologic test using Salpingitis and epididymitis often Useful in the diagnosis of Method most often used in
immunofluorescence result in higher titers than superficial psittacosis if there is a rising IgG the diagnosis of C. pneumoniae
technique infections; women generally have titer infections; a 4-fold rise in titer, a
higher titers than men single IgM titer of >1:16, or an IgG
titer of >1:512 suggests infection
Nucleic acid probe assay Method currently developed for Commercial kits not available Commercial kits not available
the diagnosis of C. trachomatis;
approximately as sensitive and specific
as the best antigen methods with a
turnaround time as short as 4 h
Polymerase chain reaction Methods currently available for Commercial kits not available Commercial assays available for
(PCR), strand displacement C. trachomatis detection; a major C. pneumoniae (and Mycoplasma
amplification (SDA), and advantage is that the test can be pneumoniae) from respiratory
transcription-mediated performed with urine specimens as specimens, with the preferred
amplification (TMA) assays well as swab specimens; it is also more specimen being a nasopharyngeal
sensitive than the other methods aspirate or throat swab;
bronchoalveolar lavage and sputum
specimens are also acceptable
Herpes simplex viruses contact, which is not limited to intercourse. Genital HSV-2 infection is much more likely to recur
can be subdivided into and have asymptomatic virus shedding than HSV-1 infection. Neonatal herpes may be acquired
HSV type 1 and type 2. when the infant comes into contact with HSV, typically through an infected birth canal (it can
Oral herpes infections, also be acquired from caregivers infected with HSV). Neonatal herpes can present as a severe dis-
which are typically seminated infection predominantly affecting the liver and lungs, as a localized CNS infection, or
present as cold sores, as a skin and mucous membrane infection.
are primarily caused by
HSV-1. Genital herpes
infections are primarily Diagnosis
caused by HSV-2. The laboratory diagnosis of HSV depends on the type of infection and specimen (see Table 5–42).
Viral culture of vesicle fluid is useful for patients who present with genital lesions. The presence
of HSV-2 infection between recurrences can be confirmed by performing type-specific serologic
tests that detect antibodies to glycoprotein G. PCR testing of CSF is superior to all other methods
for the diagnosis of CNS HSV infections.
TABLE 5–42 Evaluation for Herpes Simplex Viral Infection

Laboratory Test Positive Result
Viral culture Viral culture continues to be the primary method for the diagnosis of mucocutaneous HSV infection; the greatest
likelihood for recovery of virus for culture is when a vesicular or pustular lesion is sampled within 72 h of its
appearance, a negative result for a culture does not rule out HSV infection; PCR is far superior to viral culture
for the diagnosis of HSV infection in the central nervous system
Smear with Tzanck preparation Intranuclear inclusions and multinucleated giant cells in the Tzanck preparation (Wright–Giemsa stain) support
a diagnosis of HSV infection; the sensitivity of this test for HSV infection is approximately 65%, and, therefore,
the diagnosis of HSV infection should be supported by the results of other tests; this assay cannot distinguish
between HSV type 1, HSV type 2, and varicella zoster virus infections
Direct fluorescent antibody Direct fluorescent antibody staining of cells from skin lesions, when positive, provides rapid results; however, a
preparation negative result does not rule out infection; direct fluorescence assays can distinguish between HSV type 1 and
HSV type 2
Serologic assay for antibodies HSV-2 infection can be detected with type-specific enzyme immunoassays or immunoblots that detect antibodies
to HSV to glycoprotein G; other serologic assays cannot distinguish HSV-1 and HSV-2; a negative result does not exclude
HSV, particularly during a primary infection
Polymerase chain reaction The PCR assay is the gold standard for the diagnosis of encephalitis and meningitis from HSV infection because it
is much more sensitive than culture for detection of virus; because central nervous system or disseminated HSV
encephalitis in newborns responds to therapy if it is initiated early in the course of the disease, the diagnosis of
HSV infection by PCR using cerebrospinal fluid and/or blood is particularly important
HSV, herpes simplex virus; PCR, polymerase chain reaction.
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C H A P T E R
Toxicology
James H. Nichols, Sheila P. Dawling, and Michael Laposata 6
LEARNING OBJECTIVES
1. Define therapeutic drug monitoring, and learn when it is necessary and how
it is performed for commonly monitored drugs.
2. Describe basic pharmacokinetic principles as they relate to therapeutic drug
monitoring.
3. Identify the common drugs of abuse and how they are detected in blood,
serum, urine, and other body fluids.
4. Understand the association between occupations, industries, and exposure
to specific environmental toxins.
CHAPTER OUTLINE
Introduction 159 Antidepressants 169
Therapeutic Drug Monitoring 160 Cardiac Drugs 170
Overview of Therapeutic Drug Pain Management 170
Monitoring 160 Drugs of Abuse 172
Indications for Therapeutic Drug Overview 172
Monitoring 160 Specimen Collection and Laboratory
Pharmacokinetic Principles 162 Analysis 174
Laboratory Methods 165 Selected Drugs of Abuse 175
Specimen Collection 165 Environmental Toxins 179
Selected Commonly Monitored Drugs 165 Overview 179
Antibiotics 167 Carbon Monoxide 179
Antiepileptics 168 Lead 181
INTRODUCTION
Toxicology comprises several medical applications. The analysis of drugs in human specimens can
be conducted for clinical or legal/forensic purposes. Clinical applications include the acute man-
agement of overdose and therapeutic monitoring of drug concentrations to achieve maximum
efficacy while limiting the toxicity and side effects of medications. Forensic applications of toxicol-
ogy include analysis of drugs to provide evidence for civil and criminal court cases, to investigate
the cause of death, to deter the use of performance-enhancing drugs in athletic competitions,
and to determine operator impairment related to traffic citations and vehicle accidents. Work-
place drug testing assesses pre-employment drug abuse and on-the-job impairment. Given the
number of therapeutic drugs, drugs of abuse, and environmental toxins, as well as the variety
of diseases, signs, and symptoms associated with drug exposure and overdose, there is an array
of laboratory testing strategies. For this reason, the discussion of toxicology in this chapter will
be divided into 3 broad sections—therapeutic drug monitoring (TDM), drugs of abuse, and
environmental toxins (Figure 6–1).
159
160 CHAPTER 6 Toxicology
Therapeutic drug monitoring
TDM is the practice of measuring the concentration of a drug or its

metabolites in order to optimize the dosing of that drug to an individual
patient and/or to assess patient compliance with a dosing schedule.
TDM may be required for drugs with a narrow therapeutic index,

significant side effects, or low margin of safety. Monitoring is useful
when the therapeutic range for a drug significantly overlaps the toxic
range, when a drug cannot be dosed based on clinical observation, or
when patients have compliance problems.
Not all drugs require monitoring.
Detection of drugs of abuse
An abused drug is any compound that is consumed in greater amounts

or in a manner that is neither approved nor supervised by medical staff.
Drugs of abuse are agents used recreationally for euphoria, stimulant,
sedative, or other effects. Analysis is intended to detect past use by the
patient.
Detection of environmental toxins
Environmental toxins are potentially hazardous substances that

contaminate the air, water, or soil. Exposure to environmental toxins
may be monitored by specific tests for clinical diagnosis and treatment.
FIGURE 6–1 Considerations in therapeutic drug monitoring, drugs of abuse detection, and
detection of environmental toxins.
THERAPEUTIC DRUG MONITORING

Overview of Therapeutic Drug Monitoring
TDM is the practice of measuring the concentration of a drug or its metabolite in order to opti-
mize the dosing of that drug to an individual patient and/or to assess patient compliance with a
The goal of therapeutic dosing schedule. The goal of TDM is to improve drug efficacy—the likelihood of a therapeutic
drug monitoring is to effect while avoiding or minimizing adverse effects. Table 6–1 lists some commonly monitored
increase the likelihood of drugs. Patients do not require monitoring for most drugs. However, for a limited group of agents
a therapeutic effect and or for patients with certain conditions (for instance, limited renal function, pregnancy, newborn
avoid or minimize adverse or geriatric age groups), TDM plays an essential role in establishing the appropriate therapeutic
effects. Patients do not dosing regimen.
require monitoring for Prior to the 1960s, drug dosing was entirely empirical. For certain agents, this trial-and-error
most drugs. approach gave wide variations in patient response and a significant incidence of toxicity. Since
then, physicians have learned to optimize drug dosages and delivery while avoiding many of the
drug’s adverse effects. This has been achieved through the development of sensitive and rapid
laboratory assays and the establishment of therapeutic ranges for common medications.
Indications for Therapeutic Drug Monitoring

TDM is performed to optimize the dose of a drug to an individual patient. Drugs with a nar-
row therapeutic index or margin of safety (the difference between the effective dose and the
toxic dose) are potential candidates for therapeutic monitoring (Table 6–2). TDM is useful for
CHAPTER 6 Toxicology 161
TABLE 6–1 Commonly Monitored Drugs

Methotrexate
Immunosuppressants
• Tacrolimus (FK-506)
• Cyclosporin
• Sirolimus (rapamycin)
Antibiotics
• Gentamicin
• Tobramycin
• Vancomycin
Antiepileptics (first generation)

• Phenytoin
• Phenobarbital
• Carbamazepine
• Valproic acid
• Clonazepam
Antiepileptics (second generation)

• Lamotrigine
• Levetiracetam
• Oxcarbazepine
Tricyclic antidepressants
• Amitriptyline
• Desipramine
• Doxepin
• Imipramine
• Nortriptyline
Lithium
Cardiac agents
• Digoxin
Pain management
• Buprenorphine
• Methadone
drugs that display significant pharmacokinetic variability that may be caused by drug interac-
tions, genetic variation in drug metabolism, nonlinear kinetics, physiologic conditions such as
pregnancy and aging, and underlying diseases that alter the effective amount of drug delivered
to or metabolized by the body. When patient compliance is in question, drug monitoring may
be used to demonstrate the presence or absence of the prescribed agent. TDM requires a suit-
able laboratory assay and the establishment of a therapeutic reference range that correlates with
efficacy and/or toxicity.
TABLE 6–2 Indications for Therapeutic Drug Monitoring

The prescribed drug has a low margin of safety; that is, toxic blood drug concentrations or dosages are only
slightly greater than therapeutic ones (a narrow therapeutic index)
Patient compliance with their prescribed drug regimen is uncertain
The drug does not act via irreversible inhibition (“hit and run” effect)
Symptoms of underlying disease are difficult to distinguish from drug toxicity
The treatment goal is not an objectively measured end point (such as blood pressure)
The prescribed drug has significant pharmacokinetic variability as a result of:

• Interindividual metabolic capacity
• Nonlinear (zero-order) drug kinetics
• Frequent drug–drug interactions
• Physiologic conditions (eg, aging, pregnancy)
• Underlying disease state (eg, liver or renal impairment)
TDM is performed by measuring the concentration of a drug and metabolite(s). Blood or

serum/plasma is the usual sample for TDM, but in some cases, urine or oral fluid samples are
used to evaluate patient compliance. The most common examples of urine and oral fluid sampling
are monitoring of buprenorphine, methadone, and oxycodone for compliance. By using blood
levels to guide drug therapy, a proportional relationship is assumed between the plasma/serum
concentration, the concentration of drug at the organ cellular level, and pharmacologic effect.
For practical reasons, only blood levels of the drug are measured, because tissue concentrations
cannot be easily sampled or analyzed. This pharmacokinetic principle of homogeneity defines
the timing of sampling for TDM, since the concentrations of drug in blood at the moment of
sample collection must reflect a proportional and constant (steady-state) concentration at the
end organ and be reflective of drug effects at the cellular level. Most TDM samples are collected
as trough concentrations, the lowest level just prior to the next dose, or as peak concentrations,
30 to 60 minutes after the dose, when blood levels are most reflective of the tissue concentration
and drug efficacy or toxicity.
Pharmacokinetic Principles
Pharmacokinetics is the study of drug interaction (absorption, metabolism, and clearance) within
the body. Drug behavior in the body can be described by the LADME mnemonic. The “L” stands
for liberation or release of the drug from its dosage form. The “A” is absorption that describes
the movement of drug from the administration site into circulation. Distribution is the “D” and
describes the reversible movement of drug through the circulatory system and body tissues.
Metabolism or “M” is the chemical conversion of drug to active and inactive compounds. Finally,
the “E” indicates how the body eliminates the drug.
Drugs behave in the body based on their chemical characteristics at the molecular level.
Drugs can be acidic, basic, neutral, or polar (Table 6–3). The charge and dissociation constant
(pK) of the drug influences its absorption, distribution, and elimination characteristics. The dis-
sociation constant of the drug also affects how the drug can be extracted from patient samples and
analyzed in the laboratory.
Liberation and Routes of Drug Administration

Drugs can be delivered to the body in a variety of ways. Patients may take a drug orally
(PO) by pill (eg, aspirin) or dissolve a powder in a liquid drink (eg, laxatives). Drugs can be
TABLE 6–3 Characteristics of Chemical Groups on Drugs

Acidic drugs
pK
R−C=O < > R−C=O + H+
| |
OH O−
Unionized Ionized
Basic drugs
pK
H+ + R−NH2 < > R−NH3+
Unionized Ionized
Neutral Drugs Polar Drugs

R−CH3 R−OH
(Oil-like) (Water-like)
The chemical groups on a drug determine the drug’s characteristics and behavior in the body
Acidic and basic chemical groups on drugs are in equilibrium between the unionized (uncharged) and ionized
(charged) forms of the molecule
Unionized forms can passively diffuse while charged forms required active transport and bind to proteins
and other counterions
Neutral drugs carry no charge and can be hydrophobic, oil-like, or polar, hydrophilic and attract water
delivered intravascularly (IV) through a needle directly into circulation (antibiotics, like van-
comycin). Some medications are delivered under the tongue, sublingual (SL), like nitroglycerin
for cardiac pain or angina. Others may be injected under the skin, subcutaneous (SC), like com-
pounds in a tuberculosis test, or intramuscularly (IM), such as vaccinations. Rectal (supposito-
ries) and transdermal (eg, fentanyl pain patches) are other methods of delivering a drug. The
route of administration will affect absorption and bioavailability of the drug to the body.
Drug Absorption
The route of drug administration and the formulation of the drug affect the rate and extent of
drug absorption. For example, oral drug absorption is affected by many factors including drug
solubility in enteral fluid, the acid–base characteristics of the drug, the lipid solubility of the drug,
interferences with absorption by food, destruction of the drug by gastrointestinal flora, coadmin-
istration of other drugs—especially antacids, cholestyramine, and other resin-binding agents—
blood flow to the gastrointestinal tract, and gastrointestinal transit time. Some orally delivered
drugs are also subject to a significant “first-pass effect,” whereby they are largely metabolized
by the liver to inactive compounds before reaching the systemic circulation. IV administration
delivers drug directly into circulation bypassing “first-pass metabolism,” and the amount of drug
delivered IV is often compared with other delivery options for determining the extent or amount
of drug that is absorbed from a specific formulation. Significant variability in drug absorption is
thus a common indication for TDM.
The chemical characteristics of a drug also affect the rate and extent of drug absorption.
Acidic drugs carry a carboxyl group, R−COOH (Table 6–3). This acidic group is unionized
or uncharged at pH levels below the drug’s dissociation constant and is ionized or charged
(R−COO−) at pH levels above the drug’s dissociation constant. Drugs that act as strong acids
have dissociation constants with a pK <5, such as salicylate, penicillin, and analgesics. Strongly
acidic drugs are unionized and do not carry a charge at the acidic pH of the stomach while
they carry a charge at the more basic pH of the intestines. So, drugs like salicylate are passively
absorbed in the stomach, but require active transport for absorption across the intestines. Weak
acids (barbiturates, sulfonamides, and thiazide diuretics) have dissociation constants in the
range 5 to 11, and are preferentially absorbed in the intestines compared with the stomach.
Strongly acidic drugs tend to be fully dissociated or charged at blood pH of 7.4 while weak acids
may have significant amounts of the unionized form present in the blood. Due to the acidic pH
of urine, weak acids that are ionized at blood pH may become unionized in urine and prone to
greater reabsorption. Basic drugs contain an amine group (R−NH3). Basic groups are ionized
(R−NH2+) below and unionized (uncharged) above their dissociation constant. Basic drugs can
act as weak bases (eg, anesthetics, opiates, and antidepressants) with pK <10 or strong bases
(eg, amphetamines and bronchodilators). Basic drugs tend to be significantly ionized (charged)
at blood pH. Drugs can also be neutral and carry no charge across the range of physiologic pH.
Neutral drugs can be lipophilic and act like fats (eg, corticosteroids) or polar and hydrophilic
and attract water molecules (eg, digoxin).
Distribution
After absorption, drugs distribute throughout the body through the circulatory system, lymphatic
system, and tissue fluids. The amount of free drug available to act at organ receptors is affected
by both protein and tissue binding. Protein binding is another consideration in TDM. Binding
to plasma proteins occurs to some extent for most drugs, with bound and free (unbound) drugs
existing in equilibrium. Although it is only the free drug fraction that is biologically active, most
laboratory assays measure the total drug concentration, that is, the sum of the bound and the
unbound drugs. Several factors can cause changes in plasma proteins and, consequently, affect free
drug levels. Acid/neutral drugs tend to bind albumin while basic/neutral drugs bind α1-acid gly-
coprotein. Some drugs have specific binding proteins such as cortisol and corticosteroid-binding
globulin, also known as transcortin. These proteins serve as transport proteins for drugs from the
site of absorption to the tissue where drug can act, and as delivery mechanisms to the liver for
metabolism or the kidney for elimination. Disease alterations in protein concentration can affect
the concentration of free drug. For example, hypoalbuminemia, which occurs in the elderly and
in patients with cirrhosis, may cause an increased free drug fraction in the setting of normal total
drug levels. α1-Acid glycoprotein is an acute-phase reactant and levels of this protein increase in
acute and chronic diseases. Increases in α1-acid glycoprotein create more binding sites for drug,
so less free drug will be available in light of the same total drug concentration in a sample. The
presence of uremia in disease results in compounds binding to albumin, displacing drug from the
protein and elevating the free drug fraction. TDM can determine the proportion of free and total
drugs in disease and individualize the dosage to the patient’s condition.
Drug Metabolism
Drug metabolism typically renders nonpolar, lipophilic drugs into more polar, water-soluble
compounds for elimination. The liver is the primary site for drug metabolism. Genetic variants,
age, cirrhosis, and other hepatic conditions may adversely affect drug metabolism, and thus
predispose a patient to toxicity. Many drugs are hepatic enzyme inducers or inhibitors and thus
can influence the rate of their own metabolism, as well as the metabolism of many other drugs.
Pharmacokinetics is the Pharmacogenetics is study of drug action and metabolism based on genetics. Different genes
study of drug interaction can lead to changes in drug metabolism and produce an individualized response to a drug. Fast
(absorption, metabolism, metabolizers will change parent drug to a metabolite at a greater rate than slow metabolizers,
and clearance) within the depending on the genes expressing metabolizing enzymes in the patient. For example, patients with
body. Pharmacogenetics the slow metabolizer gene will acetylate procainamide (a cardiac drug) to N-acetylprocainamide
is the study of drug metabolite at a slower rate than fast metabolizers, and may be more prone to toxicity while on the
metabolism based on same dose of drug.
genetics.
Drug Elimination
Elimination is the removal of drug and metabolites from the body. Drug can be eliminated
through the kidneys, the liver, the lungs, the skin, the feces, and by other means. Elimination of
many polar, nonlipophilic drugs is achieved primarily through renal excretion, which is depen-
dent on adequate kidney function and renal blood flow. Other parameters relevant to elimination
through the kidneys include urine pH and the properties of the drug itself, such as the dissocia-
tion constant, pK, and molecular size. Drug clearance is the theoretical volume of serum/plasma
that is completely cleared of a drug per unit time. Importantly, clearance is the sum of all elimi-
nation mechanisms—hepatic, renal, lung, and any other—for a particular drug. Patients with
impaired drug clearance may need more frequent monitoring.
In TDM, drug levels are most often determined only after steady state has been achieved.
Steady state is the condition that occurs when the amount of drug entering the system equals
the amount being eliminated. Steady-state concentration is compared in relation to a target
range to determine changes in dosing. The target range is established from experimental dos-
ing studies to determine the optimum drug concentration where a drug is most effective while
causing the least undesirable side effects and toxicity. The target range is a generalized range
that fits most patients, but that range may need to be adjusted or altered in certain disease states
and physiologic conditions. TDM allows physicians to optimize drug dosage to a patient’s indi-
vidual situation.
Most but not all drugs Most but not all drugs are eliminated by first-order (or linear) kinetics. This means that a
are eliminated by first- constant fraction of total drug is eliminated per unit time. All drugs have a biological half-life. For
order (or linear) kinetics. drugs that follow first-order elimination kinetics, changes in dose will generally cause predict-
This means that a able changes in blood levels. Increases in drug concentration lead to increases in the rate of drug
constant fraction of drug elimination. Some drugs, however, are eliminated by zero-order (or nonlinear) kinetics, such
is eliminated per unit that a constant amount of drug is eliminated per unit time. Typically, metabolism by zero-order
time. Other drugs are kinetics occurs when elimination pathways for that drug have been saturated. Under these cir-
eliminated by zero-order cumstances, the biological half-life is not constant but depends on drug concentration. As a result,
(or nonlinear) kinetics, small increments in dose may cause disproportionately large increments in blood levels. Due to
such that a constant their lack of a predictable dose–response relationship, drugs that follow zero-order kinetics oft en
amount of drug is require monitoring.
eliminated per unit time.
Assuming first-order kinetics, 5 half-lives are required after initiation of drug therapy to
reach nearly complete (97%) steady state (5 half-life rule). Five half-lives are also required for
nearly complete clearance of a drug after the termination of therapy, and for attaining a new
steady state whenever a dosing regimen has been changed.
Drug Interactions and Dose Adjustments

Many patients may take more than 1 medication, and those drugs can interact in the patient’s
body. Drug interactions may cause displacement of bound drug from proteins. The clinical sig-
nificance of the interaction is likely to be increased when both drugs are highly protein bound
(80% or more), when 1 of the drugs has a higher binding affinity, or when 1 of the drugs is present
in higher concentration than the other. Dosing adjustments may be required in these instances.
Displacement of bound drug does not inevitably lead to an increased free drug level, because
free drug is subject to increased metabolism and elimination. Increases in plasma proteins and
drug binding may also occur as an acute-phase response or during pregnancy, and, consequently,
higher dosing may be necessary. Caution must be used when interpreting total drug levels in
patients with possible protein disturbances or drug interactions, and free drug levels may be more
useful in these situations.
Laboratory Methods
Currently, most clinical laboratories utilize immunoassays for the rapid and quantitative mea-
surement of therapeutic drugs. In an immunoassay, drug in the patient’s sample competes with a
drug conjugate (drug attached to an enzyme or fluorescein molecule) for the binding of specific
antibodies. Antibody binding results in blocking enzyme activity or in enhancing fluorescence
polarization. By measuring enzyme activity or fluorescence polarization, the amount of drug in
the patient sample is quantitated. Chemiluminescent immunoassays offering superior sensitivity
are also available for drug analysis. Other immunoassay methods such as ELISA and radioimmu-
noassay are less commonly used. More complex laboratory techniques, such as chromatography
with ultraviolet or mass spectral detection, are also commonly utilized for drug measurements.
(See Chapter 2.) Immunoassays offer advantages over chromatographic methods, because immu-
noassays can be automated and analyze a greater number of samples more rapidly with less labor
and cost. Only the total drug concentrations are routinely measured. Free drug levels require a
more time-consuming and expensive ultracentrifugation or dialysis equilibrium steps to separate
the protein-bound drug from the free drug. Free drug concentrations are typically lower than
total drug concentrations by a factor of 2- to 20-fold, so more sensitive assays are required.
Specimen Collection
The appropriate specimen for therapeutic drug measurements is usually serum or plasma. Most
laboratories do not accept gel separator tubes as the gel can bind drugs and interfere with drug
recovery. Immunosuppressant levels are measured using whole blood due to the distribution
and concentration of drug in RBCs, which are removed in the preparation of serum/plasma.
In general, trough levels
EDTA-anticoagulated whole blood is the appropriate sample for these immunosuppressant drug
are drawn just prior to the
measurements. Urine samples are frequently used to evaluate patient compliance in cases of ther- next dose and are used
apeutic administration of buprenorphine, methadone, and several opiates (including oxycodone). to evaluate the likelihood
Saliva or oral fluid may be appropriate for monitoring some medications, such as theophylline, of a therapeutic effect.
in pediatric patients or in those for whom phlebotomy is difficult. Oral fluid is also not subject to Peak levels are drawn at
adulteration or substitution, which can be an issue with monitoring for pain management compli- varying times, depending
ance in patients prone to abuse. In general, trough levels are drawn just prior to the next dose and on the particular drug,
are used to evaluate the likelihood of a therapeutic effect. Peak levels are drawn at varying times, and are used typically to
depending on the particular drug, and are used typically to assess toxicity risk. assess toxicity risk.
Selected Commonly Monitored Drugs

Selected individual drugs and considerations for TDM are presented in Table 6–4. The required
specimen volume and preservative will vary by analytical methodology, so the described collec-
tion instructions are only a guide. The reader should refer to specific instructions from the labo-
ratory. The general monitoring recommendations will depend on the motivation for monitoring
the drug, possible drug interactions, and whether the patient is stable or showing signs of toxicity.
Therapeutic ranges are only suggestions and will vary by patient, condition, and the presence of
other medications.
TABLE 6–4 Therapeutic Drug Monitoring for Commonly Monitored Drugs

Specimen
Monitoring Collection Tube Suggested Special
Drug Recommendations and Instructions Therapeutic Range Considerations
Methotrexate 24, 48, 72 h after bolus; then daily 5 mL red top; wrap in <10 μmol/L at 24 h Monitoring guidelines
until below cytotoxic levels foil to protect from <1 μmol/L at 48 h are for high-dose therapy
light; indicate time <0.4 μmol/L at 72 h (>20 mg/kg) only
past bolus
Tacrolimus Trough levels, 12 h post dose 3 mL purple top 5–20 ng/mL Cross-reactivity with
(FK-506) its metabolites in
immunoassays
Cyclosporine Trough levels, 12 or 24 h post dose 3 mL purple top; Transplant of: Ranges depend on organ
avoid drawing from (1) Liver: 400-800 ng/mL transplanted and time
line of administration (2) Heart: 150-300 ng/mL since transplant
(3) Kidney:
(a) <3 months: 150-250 ng/mL
(b) >3 months: 100-200 ng/mL
Aminoglycosides Peak: 5 mL red top Gentamicin—peak: 5-10 μg/mL, Guidelines for conventional
(1) IV: 30-60 min post dose trough: <2.0 μg/mL dosing only (not low-dose
(2) IM: 60-90 min post dose Tobramycin—peak: 4-8 μg/mL, therapy or pulse therapy)
Trough: 30 min prior to next dose trough: <2.0 μg/mL
Vancomycin Either peak or trough, once per day 5 mL red top Peak: 30-40 μg/mL, trough: Frequency of monitoring
5-10 μg/mL dependent on clinical
situation
Phenytoin Peak for toxicity is 4-5 h after dose; 5 mL red top 10-20 μg/mL Pertains to assay that
trough for monitoring measures total drug
(free + bound)
Phenobarbital Trough 5 mL red top 15-50 μg/mL Steady state attained

in 2-3 weeks
Carbamazepine Peak level for toxicity is 2-4 h after 5 mL red top 4-12 μg/mL Not helpful for
dose; trough for monitoring idiosyncratic toxicities
Clonazepam Peak for toxicity is 4 h after dose; 1 mL red or green top 20-60 μg/mL
trough for monitoring
Lamotrigine Peak for toxicity is 2-4 h after dose; 1 mL red or green top 3-14 μg/mL
Levetiracetam Peak for toxicity is 1 h after dose; 1 mL red or green top 5-30 μg/mL
Oxcarbazepine Peak MHD for toxicity is 4-6 h after 1 mL red or green top 15-35 μg/mL MHD
dose; trough for monitoring
Valproic acid Trough is not well defined 5 mL red top 50-100 μg/mL Upper limit of therapeutic
range
Tricyclic Steady state occurs in about 5 days; 5 mL red top a

Amitriptyline: 120-250 μg/L Measure sum of parent
antidepressants 10-14 h after once per day dosing; Desipramine: 150-300 μg/L and active metabolite for
4-6 h after twice per day dosing a
Doxepin: 150-250 μg/L drugs noted with “a” in box
a
Imipramine: 150-250 μg/L at left
Nortriptyline: 50-150 μg/L
Lithium 10-14 h after dose; then biweekly or 5 mL red top 0.5-1.5 mmol/L (avoid green-top Toxicity may occur at
weekly until steady state; then every tubes) <1.5 mmol/L, especially in
1-3 months patients who show chronic
toxicity
Digoxin 8 h after PO dose; 12 h after IV dose; 5 mL red top 0.9-2.0 ng/mL Specimen collection time
and at steady state (1 week after is crucial to avoid falsely
initiation) high levels; STAT levels
occasionally necessary
IM, intramuscular; IV, intravenous; PO, oral; MHD, monohydroxy carbazepine.
Measure the sum of parent and active metabolite, that is, (amitriptyline + nortriptyline), (imipramine + desipramine), and (doxepin + desmethyldoxepin).
a
Methotrexate
Methotrexate is a folate antagonist used in the treatment of a wide variety of neoplasms.
Dose-related toxicity is common with high-dose methotrexate therapy (defined as >1 g/m2
or 20 mg/kg). Adverse effects include immunosuppression, and diverse organ damage including
renal failure, myelosuppression, hepatic toxicity, neurotoxicity, gastrointestinal toxicity, and death.
Toxicity correlates with serum methotrexate concentration and duration of exposure. Patients
with poor hydration, renal insufficiency, pleural effusion, ascites, or gastrointestinal obstruction
are at increased risk for toxicity. Adverse effects of methotrexate are ameliorated by administra-
tion of leucovorin, a reduced folate. Serial methotrexate levels are used to guide the appropriate
dosing and duration of leucovorin rescue following high-dose methotrexate administration.
Immunosuppressants
The immunosuppressant drugs, tacrolimus (FK-506), cyclosporin, and sirolimus (rapamycin),
are drugs used to prevent rejection in organ transplantation. Cyclosporin is also utilized to treat
psoriasis, chronic autoimmune urticaria, and rheumatoid arthritis. These drugs were originally
discovered in bacteria (tacrolimus and sirolimus) and fungus (cyclosporin) from soil samples.
Monitoring is indicated because these drugs have a narrow therapeutic index and highly vari-
able pharmacokinetics. Adverse effects include nephrotoxicity, hepatotoxicity, pulmonary toxic-
ity, neurotoxicity (light sensitivity, tingling in the palms, and tinnitus), tremor, and hypertension.
Whole blood is the preferred specimen for TDM, as the immunosuppressant drugs con-
centrate into erythrocytes more than the plasma/serum portion of blood. Low trough concen-
trations may indicate subtherapeutic immunosuppression and can be associated with increased
risk of rejection. High trough concentrations cause increased toxicity including nephrotoxicity
that can be particularly challenging to diagnose in renal transplant patients. Drug levels must be
interpreted in conjunction with other laboratory test results and clinical findings to discriminate
between toxicity and rejection. For renal transplant patients on cyclosporin therapy, the only
definitive method for differentiating graft rejection from drug-induced nephrotoxicity is renal
biopsy. These drugs are sometimes used in combination, and with mycophenolic acid, to enhance
the immunosuppressant effects and decrease the dose and side effects.
The immunosuppressants are extensively metabolized by the liver to a number of metabolites,
some of which have immunosuppressant activity. Some metabolites can cross-react in laboratory
immunoassays, thus overestimating parent drug concentrations in situations where elimination is
impaired and when metabolites accumulate, as in cholestasis. Patients who have received mouse
monoclonal antibody therapies may also have inaccurate immunoassay results. HPLC with tan-
dem mass spectrometry is increasingly being used for laboratory analysis to circumvent cross-
reactivity with the immunoassays.
Antibiotics
Aminoglycosides
Gentamicin, tobramycin, and amikacin are aminoglycoside antibiotics. Ototoxicity and nephro-
toxicity from aminoglycosides are related to dose and duration of exposure. Numerous factors,
such as renal and cardiac function, age, liver disease, and obesity, affect the pharmacokinetic
properties of aminoglycosides. Because of the many patient factors, as well as the low margin of
safety and high incidence of dose-related toxicity, aminoglycoside levels are usually indicated in
conjunction with renal function monitoring to minimize toxicity. In patients with normal renal
function and without underlying disease, the indication for drug monitoring is less well defined.
Vancomycin
Vancomycin is a tricyclic glycopeptide antibiotic with significant dose-related nephrotoxicity
and ototoxicity. The practice of measuring vancomycin levels emerged from the guidelines for
aminoglycoside monitoring. However, the necessity for vancomycin monitoring is controver-
sial, because a good correlation between serum vancomycin levels and efficacy or toxicity has
yet to be definitively demonstrated. Adult patients with normal renal function may not require
routine monitoring. Indications for monitoring include impaired or changing renal function,
concomitant use of nephrotoxic drugs, altered volume of distribution (as in a burn injury vic-
tim), prolonged vancomycin use, higher than usual doses, and use in neonates, children, pregnant
women, and patients with malignancy.
Antiepileptics
Antiepileptics are Antiepileptics are frequently monitored to establish the dose necessary to reduce the frequency
frequently monitored and magnitude of seizures. Trough levels are used to establish minimum effective dose. When
to establish the dose toxicity is suspected, peak or random levels may be obtained. Too low a level will lead to break-
necessary to reduce the through seizures, while too high a dose can induce seizures. A therapeutic level maintains seizure
frequency and magnitude control and avoids side effects. The concentration of drug in the blood also may be used to evalu-
of seizures. ate patient compliance, and explain seizures that are refractory to drug treatment.
Phenytoin (Dilantin®)
Phenytoin (or its prodrug phosphenytoin) is a widely used anticonvulsant with nonlinear kinet-
ics and wide interindividual variability in dose requirement. Phenytoin toxicity includes ataxia,
tremor, lethargy, seizure exacerbation, and neuropsychiatric changes. Phenytoin use in certain
populations requires special consideration. Neonates and the elderly have decreased clearance.
On the other hand, children metabolize phenytoin more rapidly than adults, and, therefore, dose
adjustment is necessary at various ages. Careful monitoring in pregnancy is required due to meta-
bolic and volume changes that occur during pregnancy. Phenytoin is highly protein bound, and
conditions such as renal failure, liver disease, burn injury, and age will affect the amount of free
drug by altering the amount of plasma protein.
Extensive protein binding also predisposes phenytoin to significant interactions with other
protein-bound drugs, such as valproic acid. Coadministration of valproic acid and phenytoin is
common and may cause a decrease in total phenytoin. Valproic acid displaces phenytoin from
albumin, which causes a transient increase in free phenytoin, but this free phenytoin is read-
ily metabolized and cleared. The overall effect is usually a decrease in total phenytoin with an
unchanged level of free phenytoin. Monitoring of total phenytoin levels is sufficient for patient
management, and free phenytoin levels are not usually necessary except in renal or hepatic dis-
ease, conditions that would affect total protein or body clearance.
Phenobarbital and Primidone (Mysoline®)

Phenobarbital and primidone are used to treat all types of seizures except absence (petit mal)
seizures. The major active metabolite of primidone is phenobarbital. Clearance of both primi-
done and phenobarbital is prolonged in neonates, the elderly, and patients with hepatic and renal
dysfunction. Phenobarbital is a potent hepatic enzyme inducer, and may affect the metabolism
and levels of many other drugs metabolized by the same enzymes. Concurrent valproic acid use
significantly decreases phenobarbital clearance.
Carbamazepine (Tegretol®)
The anticonvulsant carbamazepine is used not only for seizures but also for treatment of trigemi-
nal neuralgia and bipolar disorder. Monitoring of carbamazepine levels is useful due to its slow
and unpredictable absorption. Age and hepatic function affect drug clearance. Dose-related toxic
effects include blurred vision, paresthesias, ataxia, nystagmus, and drowsiness. Carbamazepine is
metabolized to the active metabolite, carbamazepine 10,11-epoxide. Children are known to accu-
mulate the epoxide metabolite and, as a result, may present with toxicity in the setting of nonel-
evated carbamazepine levels. With chronic therapy, carbamazepine induces its own metabolism,
and dosing adjustment becomes necessary.
Valproic Acid (Depakane®, Depakote®)

Valproic acid is used to treat all types of seizures. It is also used in the treatment of migraines and
bipolar disorder. Valproic acid has a narrow therapeutic index. Dose-related adverse effects involve
primarily central nervous system (CNS) depression. The average half-life of valproic acid is about
12 to 16 hours, but there is significant interindividual variability, and use of a sustained-release
formulation is popular. The half-life of valproic acid is prolonged in neonates and in patients
with liver dysfunction. Extensive protein binding accounts for the increased valproic acid toxicity
observed in patients with uremia and cirrhosis.
Second-generation Antiepileptics
The second-generation antiepileptics encompass a range of drugs with different chemical struc-
tures and pharmacokinetics. Some are protein bound (lamotrigine is 55% bound to albumin)
while others are not (levetiracetam is <10% protein bound). Common adverse effects include diz-
ziness, ataxia, nausea, and vomiting. Decreased hematocrit and neutropenia can also be seen with
lamotrigine. In general, the second-generation antiepileptics have a wider therapeutic index and
fewer side effects than the first-generation drugs. HPLC and immunoassays are available. How-
ever, therapeutic and toxic ranges have not been established for all of these drugs. So, monitoring
is generally conducted to define the individual level at which a patient is achieving therapeutic
action with fewest side effects for future reference, for compliance, and for documentation of the
level at which side effects are evident for that patient.
Antidepressants
Tricyclic Antidepressants
Tricyclic antidepressants are monitored for multiple reasons. There is significant interindividual Tricyclic antidepressants
variation in metabolism and elimination, such that standard dosing results in therapeutic levels in are monitored for
less than half of patients. Genetic variation accounts for some of this variability. The fraction of multiple reasons. There is
“poor metabolizers” is about 17% of Caucasians and 5% of other ethnic groups. Other indications for significant interindividual
monitoring include a narrow therapeutic index, multiple drug interactions, and patient compliance. variation in metabolism
Tricyclic antidepressants have a low margin of safety and cause anticholinergic toxicity, sei- and elimination, such
zures, and arrhythmias in overdose. Although the correlation between toxicity and blood level that standard dosing
is poor, there are general guidelines. Levels in excess of 500 μg/L may be associated with anti- results in therapeutic
cholinergic toxicity (flushing, tachycardia, fever, dilated pupils, dry mucous membranes, urinary levels in less than half of
patients. Genetic variation
retention, and absent bowel sounds). Cardiotoxicity is more likely to occur at levels greater than
accounts for some of this
1000 μg/L in acute overdose.
variability.
Lithium
Lithium is a univalent cation most commonly used to treat bipolar disorder. Lithium monitor-
ing is useful due to its narrow therapeutic index and the wide interindividual variation in dose
requirement.
Excretion of lithium is primarily renal. Children have increased clearance, while the elderly
have decreased clearance. Lithium excretion parallels sodium excretion. Therefore, patients on
stable doses of lithium may become toxic in states of sodium conservation such as fever, excessive
sweating, lack of fluid intake, and diarrhea.
Toxicity is usually associated with levels in excess of 1.5 mmol/L. However, toxicity may
occur at lower levels, especially in cases of chronic toxicity. Lithium overdose is characterized by
lethargy, weakness, slurred speech, ataxia, tremor, and myoclonic jerks. Severe toxicity may result
in seizure, hyperthermia, and coma. Management of patients who have ingested sustained-release
lithium preparations is difficult, and serum measurements play a crucial role in the decision to
instigate hemodialysis or whole bowel irrigation. Analytical methods involve the use of ion-
specific electrodes, and spectrophotometry or colorimetric tests.
Later-generation Antidepressants
Fluoxetine was the first selective serotonin-reuptake inhibitor used to treat depression. Fluoxetine
monitoring is useful when patient compliance is in question. Further monitoring is not likely
to be beneficial since fluoxetine has a wide therapeutic index, and there is a poor correlation
between blood levels and clinical response. Fluoxetine is metabolized by the liver to the active
metabolite norfluoxetine.
Other serotonin-reuptake inhibitors/later-generation antidepressants—such as sertraline
(Zoloft®), paroxetine (Paxil®), fluvoxamine (Luvox®), citalopram (Celexa®), quetiapine (Seroquel®),
trazodone (Deseryl®), and venlafaxine (Effexor®)—do not require routine monitoring due to their
wide therapeutic indices.
Cardiac Drugs
Digoxin
Digoxin is a commonly used drug in the treatment of heart failure and arrhythmias, and it has
a low therapeutic index. There is significant interindividual variation in digoxin absorption and
distribution along with prolonged clearance in patients with impaired renal function. Digoxin
overdose is characterized by gastrointestinal distress, confusion, visual changes, hyperkalemia,
and life-threatening cardiac toxicity. Overdoses may be treated with an antidigoxin antibody
antidote. Such treatment typically renders subsequent blood digoxin concentrations unreliable.
Blood digoxin immunoassays are generally less reliable than immunoassays for other therapeutic
agents. Interferences with digoxin immunoassays are frequently reported. These interferences are
referred to as digoxin-like immunoreactive substances (“DLIS”).
Pain Management
Acetaminophen
When it is used in the Acetaminophen is a therapeutic drug used as an analgesic and an antipyretic. When it is used in
recommended doses, it is the recommended doses, it is not necessary to measure acetaminophen levels. However, excess
not necessary to measure intake of acetaminophen can be associated with severe liver injury. Thus, acetaminophen is a
acetaminophen levels. representative of many compounds with a wide therapeutic window that does not require thera-
However, excess intake peutic monitoring when used in recommended doses. However, because toxicity can occur if the
of acetaminophen can be upper limit of the window is exceeded, monitoring acetaminophen levels in patients with excess
associated with severe intake is critical, particularly since an antidote to the major toxic effect can be administered.
liver injury. Table 6–5 presents an overview of the laboratory evaluation for acetaminophen toxicity. Immu-
noassays are available for the rapid determination of serum/plasma levels.
Acetaminophen is rapidly absorbed from the gastrointestinal tract. The plasma concentra-
tion reaches its highest level 30 to 60 minutes after a dose. One of the compounds resulting from
acetaminophen metabolism is an oxidation product that is hepatotoxic. Normally this metabolite
is detoxified by binding to glutathione in the liver. With excess intake of acetaminophen, the pro-
duction of the toxic metabolite exceeds the amount of hepatic glutathione, and this permits the
toxic metabolite to produce liver injury. Renal damage also may occur as a result of injury by the
same compound.
TABLE 6–5 Laboratory Evaluation for Acetaminophen Toxicity

Laboratory monitoring of The importance of laboratory monitoring is related to the use of

acetaminophen concentration N-acetylcysteine as a treatment for the acetaminophen overdose; it is
important that the neutralizing effect of N-acetylcysteine be provided before
acetaminophen metabolites produce liver injury. To determine whether the
acetaminophen ingestion is likely to cause liver toxicity, a 4-h postingestion
serum concentration should be obtained; the serum concentration of the
drug will be used to determine if the patient is likely to experience liver
injury and, if so, treated with N-acetylcysteine. If the first acetaminophen
level is obtained more than 4 h post ingestion, a nomogram can be used
(available in many textbooks) to determine if the acetaminophen level at
that time post ingestion is likely or not likely to be associated with liver injury
Liver function tests Hepatic necrosis becomes evident 24-48 h after the ingestion of the excess
amount of acetaminophen if the patient is not treated; at that time, standard
liver function tests such as AST (SGOT), ALT (SGPT), bilirubin, as well as the
prothrombin time, can be used to assess the extent of liver injury
ALT, alanine aminotransferase; AST, aspartate aminotransferase. The prothrombin time is a good prognostic tool when used as an
indicator of hepatic recovery.
The recommended daily dose of acetaminophen is no more than 4 g per day. A single dose of The recommended daily
10 to 15 g may produce liver injury. Fatal disease is usually associated with ingestion of ≥25 g of dose of acetaminophen
acetaminophen. Acetaminophen at slightly more than the recommended 4 g per day can produce is no more than 4 g per
hepatotoxicity when the patient has also ingested ethanol, and this response can be exacerbated day. A single dose of 10
if the patient had been fasting prior to ingestion of acetaminophen and ethanol, or takes another to 15 g may produce liver
enzyme-inducing drug such as phenytoin. The ingestion of acetaminophen at greater than recom- injury. Fatal disease is
mended doses produces corresponding elevations of acetaminophen in the blood, and the level of usually associated with
the drug in the blood correlates with the severity of hepatic injury. ingestion of ≥25 g of
Acute manifestation of excess acetaminophen intake typically occurs 2 to 3 hours after inges- acetaminophen.
tion. Most often this includes nausea, vomiting, and abdominal pain. Cyanosis of the skin and
fingernails may be observed as a result of methemoglobin generation from the overdose. The full
extent of liver damage usually becomes apparent 2 to 4 days after drug ingestion. At that time,
liver function test results, including the prothrombin time, become abnormal. A variety of associ-
ated abnormalities, including electrolyte disturbances, can occur if there is significant liver dam-
age. Acute renal failure also may occur, even if liver failure is not observed.
Aspirin
Aspirin (acetylsalicylic acid) is a therapeutic drug in use for more than a century as an analge-
sic, antipyretic, anti-inflammatory, and antithrombotic agent. It is readily absorbed and rapidly
metabolized by hydrolysis to salicylic acid. Peak concentrations occur within 1 to 2 hours with a
therapeutic dose. Between 50% and 90% is bound to albumin in a dose-dependent manner. Fur-
ther metabolism produces salicyluric and gentisic acids and glucuronide conjugates that are renally
excreted. Aspirin is contained in many preparations, including those with other analgesics. When
used in therapeutic doses, it is not necessary to measure levels. However, chronic salicylate poison-
ing (salicylism) carries a high morbidity (30%) and mortality (25%), and is difficult to diagnose
without monitoring levels since the patient may be too confused to give a reliable history. Table 6–6
presents an overview of the laboratory evaluation for salicylate toxicity. Immunoassays are available
for the rapid determination of serum/plasma levels. About 500 mg/kg as an acute dose is potentially
lethal in comparison to a normal dose of 15 mg/kg. When taken in therapeutic doses, the half-life
TABLE 6–6 Laboratory Evaluation for Aspirin Toxicity

Detection of aspirin metabolites The importance of these tests is to establish the diagnosis of poisoning.
in urine by color test (Trinder Since a number of preparations are available containing sustained-release
reagent); monitoring of serum aspirin, it is recommended that serial blood samples be drawn at 3-h
salicylate concentration by intervals to determine whether the drug concentration is still rising
enzymatic assay or immunoassay
The Done nomogram interprets the serum salicylate concentrations taken
at 6 h after acute ingestion as follows:
• <50 mg/dL: asymptomatic
• 51-110 mg/dL: mild-to-moderate toxicity
• >110 mg/dL: serious toxicity
The use of the Done nomogram is unreliable when:

• There has been a previous ingestion within 24 h
• Poisoning is chronic (concentrations >30 mg/dL indicate serious toxicity)
• Enteric-coated or sustained-release preparations have been ingested
• Renal failure is present
Treatment for aspirin overdose is symptomatic and supportive—

administration of repeat doses of oral activated charcoal may be given in
an attempt to prevent further absorption and increase fecal elimination.
Bicarbonate is used to counteract the metabolic acidosis, and calcium
and electrolytes are administered to prevent seizures and cardiac failure.
Hemodialysis may be indicated at concentrations above 100 mg/dL
(>40 mg/dL in chronic salicylism), and to support renal function and
electrolyte balance
Regular monitoring of renal function, blood gas and lactate, and

coagulation assessment are important for patient care
is 2 to 5 hours, but metabolism becomes saturated once the dose exceeds about 30 mg/kg, causing a
delay in drug elimination. An early feature of toxicity is respiratory alkalosis through direct stimu-
lation of the respiratory drive center, followed by vomiting. The latter mechanism of toxicity results
from uncoupling of oxidative phosphorylation, leading to ketosis, metabolic acidosis, and pyrexia,
with further dehydration and electrolyte imbalance. Hematologic consequences arise that manifest
as an increased prothrombin time, GI bleeding, and occasionally DIC.
Other Pain Management Drugs

Buprenorphine and methadone are analgesics that are commonly utilized for opiate withdrawal,
but have found recent medical application in the management of chronic pain. Fentanyl and oxy-
codone are other drugs utilized in pain management that may be monitored. Serum/plasma levels
of these drugs correlate poorly with clinical effect because of tolerance. The safety of these drugs
in doses utilized for pain management does not typically require monitoring and dosing can be
adjusted based on pain relief. However, these drugs have high abuse potential, so urine tests are
sometimes used to monitor for compliance and ensure that the patient is not diverting the drug
for sale or other purposes. Immunoassays are available for analysis of these drugs in urine samples.
DRUGS OF ABUSE
Overview
Drug of abuse testing Drug of abuse testing (DAT) includes testing for the use of illicit drugs (eg, cocaine and phen-
(DAT) includes testing cyclidine [PCP]) and potentially addictive or harmful therapeutic agents (eg, benzodiazepines,
for the use of illicit drugs, opiates, and amphetamine). The goal of DAT is to detect past exposure or use of a drug. Quantita-
and potentially addictive tive levels of a drug or its metabolite in fluids are not required. Laboratory analysis determines
or harmful therapeutic if a drug is above (ie, “present”) or below (absent) a defined cutoff concentration in the sample.
agents. DAT assays are designed to detect either the parent drug or metabolite with a longer half-
The goal of drug of abuse life. Cocaine, for example, has a blood half-life of less than 60 minutes, but the principal metab-
testing is to detect past olite, benzoylecgonine, has an 8-hour half-life. By detecting the metabolite, cocaine abuse can
exposure or use of a drug. be detected for several days after use as opposed to the parent drug that is cleared in less than
Quantitative levels of a 5 hours. Table 6–7 lists the typical detection window for analysis of common drugs of abuse.
drug or its metabolite in Drugs and their metabolites are much more concentrated in urine than in serum. Urine is the
fluids are not required. specimen of choice for DAT testing because of its ready availability. Other specimens have also
Laboratory analysis been utilized for DAT. Meconium (an infant’s first bowel movement after birth) has been utilized
determines if a drug is to detect in utero exposure of a fetus to drugs. Since meconium is made during the last trimester
above (ie, “present”) or of fetal development, drug exposure can be detected from as early as the sixth month of gestation.
below (absent) a defined Hair and nails have also been analyzed in attempts to detect drug use over a longer time frame
cutoff concentration in than urine, but the clinical utility of specimens other than urine remains controversial.
the sample.
TABLE 6–7 Detection Window for Commonly Abused Substances

Urine DAT Name Time (Days) Comments
Amphetamines 2-4
Barbiturates 1 to >5 Depends on barbiturate
Benzodiazepines 2 to >8 Depends on benzodiazepine
Cocaine metabolite 2 to >7 Heavy users may remain positive for 6-10 days using
sensitive immunoassays with a 150 ng/mL cutoff
Methadone 1-4
Opiates 2 to >5 Heavy users may remain positive for up to 7-8 days
Phencyclidine 7-14
Propoxyphene 2-8
THC (marijuana) 20-30

Analysis for multiple drugs in a patient’s sample can be labor intensive and expensive. Labo-
ratory analysis for drugs of abuse thus takes a 2-tier approach for efficiency. A simple, rapid assay
that can be readily automated is used to first screen a sample for the presence of a class of drugs.
These screening tests are sensitive and designed to detect a broad range of similar drugs that share
a common chemical structure. Unfortunately, screening tests are generally not very specific and
subject to a variety of cross-reactivities that can lead to false-positive test results. Thus, a second
tier of more specific testing is conducted to “confirm” the presence of a particular drug in samples
that screen positive. Common screening tests are immunoassays that can be readily performed
on chemistry instrumentation in a clinical laboratory or on point-of-care devices for testing in a
variety of settings outside of a formal laboratory. Table 6–8 lists the cutoffs and other characteris-
tics of immunoassay tests. Most immunoassays detect a number of drugs within a class of agents,
but some immunoassays are specific to a given compound, like the metabolite of cocaine, PCP,
oxycodone, and 6-monoacetylmorphine.
TABLE 6–8 Characteristics of Immunoassay Tests for Drugs of Abuse (DAT)

Typical Cutoff
Drug Class (Range) Level Causes of False- Common Drugs
DAT Name Specificity Targeted (ng/mL) positives Typically Detected Comments
Primary tests
Amphetamines Class Amphetamines 500 (300-2000) Isometheptene Amphetamine, Pseudoephedrine

Heptaminol methamphetamine no longer interferes
Seligeline MDMA (“ecstasy”) with current
Propylhexedrine immunoassays
Barbiturates Class Barbiturates 200 (100-500) — Butalbital, barbital,
secobarbital,
phenobarbital
Benzodiazepines Class Benzodiazepines 200 (100-300) Oxaprozin Diazepam, Many assays are
chlordiazepoxide, insensitive to
alprazolam, clonazepam and
oxazepam lorazepam
Cocaine Compound Cocaine 150 (100-300) — Cocaine Actual false-positives

metabolites metabolites are quite rare,
despite information
on the Internet
Opiates Class Morphine 300 (300-2000) Quinoline Heroin, morphine, Oxycodone and
and related antibiotics codeine, oxymorphone are
compounds hydromorphone, poorly detected.
hydrocodone Methadone use
does not cause a
positive test
Oxycodone Compound Oxycodone 100 (100-300) — Oxycodone, Used to assess

oxymorphone compliance/
diversion and/or
cause of a positive
opiate test
6-Monoacetylmorphine Compound Heroin 10 — Heroin use Specific for heroin

metabolite use, negating the
“poppy-seed”
defense. Used to
assess cause of a
positive opiate test
Phencyclidine Compound Phencyclidine 25 (10-25) Dextromethorphan, Phencyclidine

tramadol
THC Class Cannabinoids 50 (20-100) See comments Marijuana and Nexium use may
hashish use cause false-
positives with some
immunoassays
The second-tier or “confirmatory” testing employs a chromatographic separation prior to

mass-spectrometric detection to exactly identify the drug or metabolite in samples that gen-
erated a positive immunoassay screen. (See Chapter 2.) Confirmatory testing is expensive and
time-consuming, but is absolutely required for forensic, legal, pre-employment, and other applica-
tions of the test result that mandate definitive analysis. Confirmatory testing can take several days
and will not assist in the immediate care and management of a trauma or acute overdose patient.
The lack of confirmatory testing on clinical samples can lead to misinterpretation of test
results unless the clinician maintains an active dialogue with the laboratory regarding the likely
causes of false-positive screening tests. Many laboratories append a comment to warn that posi-
tive urine immunoassay results have not been confirmed. Common causes of false-positive test
results may also be listed with the result.
Common drugs that cause cross-reactivity and false-positive DAT results are listed in
Table 6–8. Cross-reactivity and the causes of false-positives are dependent on the antibody
specificity employed in the immunoassay, so cross-reactivities will vary from 1 manufacturer to
another and between laboratories based on the method utilized for analysis. Clinicians should
be familiar with the characteristics and limitations of the drug tests employed to analyze patient
specimens.
Drugs of abuse have no reference or therapeutic range, because the drug should not be
detected in the patient’s sample. However, sometimes a positive drug test is desired by the order-
ing physician. Urine oxycodone, for example, is utilized for pain management, but can also be
sold by a patient for monetary profit. A positive urine oxycodone test is targeted to assure that
patients are compliant with their drug regimen and not diverting the drug for other purposes.
Screening for drugs of Screening for drugs of abuse does not detect every possible drug that the patient may have
abuse does not detect ingested. Laboratory analysis is limited to the specificity of the tests at hand. Patients may have
every possible drug that access to a wide variety of plants that contain toxins or medications for which the laboratory does
the patient may have not test. In general, drug analysis is designed for rapid identification that may permit effective
ingested. Laboratory treatment (eg, acetaminophen and its antidote) to reduce the toxic effects of the compound. Con-
analysis is limited to comitant ingestion of different drugs is very common and may play a role in laboratory analysis
the specificity of the for drugs of abuse. For example, ethanol and cocaine are often ingested together, and patients
tests at hand. ingesting these drugs together can form a toxic metabolite called cocaethylene.
Specimen Collection and Laboratory Analysis

The potential for sample adulteration is a concern for any DAT. False-negative results can be
caused by purposely “adulterating” a urine sample to prevent the immunologic or indicator reac-
tion from working, leading to a false-negative result despite drug in the sample. Common house-
hold chemicals such as bleach, vinegar, sodium bicarbonate, Drano, soft drinks, or hydrogen
peroxide may be added to a urine sample in an effort to cause false-negative results. Many of these
additives work by changing the sample pH, to denature the antibody proteins used in the assay or
to shift the pH away from optimum assay conditions. Such adulteration can be detected by check-
ing the pH of urine samples submitted for drug testing. Other adulterants include glutaraldehyde
or nitrites, and can be analyzed using specific tests for adulteration in the laboratory.
Patients can take diuretics to temporarily enhance elimination. Diuretics cause an increase
in urine output, so drugs that may be present will be diluted below the assay cutoff concentration
in the urine sample. Alternatively, patients may submit water as their urine sample, so as to avoid
detection of drug use. Collection facilities can deter sample dilution by monitoring the tempera-
Sources of drug-free
ture of samples just after collection. Samples outside a physiologic temperature range should be
urine are readily available
suspected of dilution. Facilities can also cap hot water faucets and add bluing agents to the toilet
via the Internet, and
these materials are used water as additional deterrents. Laboratories can test for urine osmolality and look for specimen
by patients trying to dilution by analysis of urine creatinine.
avoid detection of their Patients may also try to avoid detection by substituting specimens. Submission of someone
drug use. In nearly all else’s urine as a patient sample is perhaps the hardest for laboratories to detect. Sources of drug-
standard tests, these free urine are readily available via the Internet, and these materials are used by patients trying
materials act like normal, to avoid detection of their drug use. In nearly all standard tests, these materials act like normal,
unadulterated human unadulterated human urine. Without close monitoring of a patient during urine sample collec-
urine. tion, sample substitution is nearly undetectable by laboratory methods.
When evaluating a patient for drug use, collecting an appropriate specimen is an important
step to detecting the presence of a drug.
• Most drugs and metabolites are concentrated in urine after use. Urine is appropriate
for qualitative analysis (presence/absence of drug) and for determining recent use of
amphetamines, benzodiazepines, barbiturates, cannabinoids, cocaine metabolites,
opiates, and their metabolites (including codeine and morphine), oxycodone, and PCP.
Urine is easy to collect and noninvasive, but subject to adulteration, dilution, and sample
substitution. Monitoring of urine collection is intrusive of patient privacy, so facilities
collecting urine samples should take steps to deter adulteration (capping hot water faucets
and using bluing agents in the toilets) and have patients remove coats and bulky clothing,
and keep purses and backpacks out of the bathroom during collection.
• Serum/plasma and blood samples provide a single time point of drug in the patient’s
system. Since blood is in equilibrium with tissue and organ receptors at steady state,
quantitative blood levels of drug can assess for intoxication and toxicity. Serum, plasma,
and blood concentration are useful for analyzing alcohol intoxication, management of
analgesic overdose (including acetaminophen and salicylates), and evaluation of TDM side
effects and toxicity. Collection of blood requires a needlestick and phlebotomy, but is not
subject to sample adulteration like urine samples.
• Other body fluids may prove useful: meconium for evaluation of in utero exposure to
drugs, vitreous humor for postmortem examination, hair and nails for past exposure to
certain drugs of abuse and toxins, and sweat or oral fluid that can be collected without
invasion of privacy and are less prone to adulteration.
It is essential that specimens are collected at an appropriate time following ingestion and that
the sample is properly preserved. Serial measurements over time may be necessary because of
delayed gastric emptying or prolonged absorption from sustained-release preparations in over-
dose cases. Blood levels do not necessarily correlate with the severity of toxicity because many
compounds distribute into specific body compartments or cells, and are therefore less detect-
able in blood. The detection of drugs in urine will depend on the patient’s renal function and
urine output, the time since last dose, chronic use of the drug, the patient’s hydration state, and
metabolism. Thus, urine collection within the first several hours after use has a better chance of
detecting drug than urine collected several days after use. Not all drugs are equally stable in a
body fluid. Alcohols should be collected anaerobically, as they are volatile compounds that will
dissipate when exposed to air. Appropriate steps should be taken to preserve the drug in the speci-
men after collection prior to analysis.
Selected Drugs of Abuse

Amphetamines
Amphetamines are stimulants and common class of abused drugs. Methamphetamine (crank,
speed), 3,4-methylenedioxymethamphetamine (a derivative of methamphetamine, also
known as MDMA or ecstasy), and several other amphetamine derivatives are used orally
and intravenously as illicit drugs. A smokable form of methamphetamine is known as “ice.”
Amphetamine-like drugs also can be used as prescription medications for treatment of a
variety of conditions and disorders. These include weight loss, narcolepsy, attention deficit
disorders, and sinus congestion. Amphetamine-like drugs work primarily by activating the
sympathetic nervous system via the CNS. Drugs in this class can produce toxicity at levels
only slightly above the usual doses, but a high degree of tolerance can develop after repeated
use. Patients who are intoxicated with amphetamine-like drugs present with CNS effects that
can extend from euphoria to seizure and coma. More severe signs and symptoms are usu-
ally associated with greater amounts of drug ingestion. The acute peripheral manifestations
extend from sweating and tremor to myocardial infarction, even if the coronary arteries are
normal. Death in amphetamine users can be caused by ventricular arrhythmia. The ingestion
of amphetamines and related drugs can be conclusively established by identification of these
compounds in urine or in gastric samples. Quantitative serum levels often do not correlate
with the severity of the signs and symptoms.
Barbiturates
Barbiturates are used clinically as hypnotic and sedative agents, for induction of anesthesia, and
for treatment of epilepsy. Ultrashort-, short-, intermediate-, and long-acting barbiturates have
different pharmacokinetic properties. All barbiturates cause a generalized depression of neuronal
activity in the brain. The toxic dose of barbiturates depends on the specific barbiturate used, the
route and rate of administration, and individual patient tolerance. Toxicity is likely to appear
when the dose administered exceeds 5 to 10 times the hypnotic dose, but chronic use may result in
marked tolerance. The patient with mild-to-moderate intoxication of barbiturates often presents
with lethargy, slurred speech, nystagmus, and ataxia. With greater amounts of drug ingestion in
overdose, hypotension, coma, and even respiratory arrest can occur. Barbiturates can be detected
in both urine and serum to document their ingestion.
Benzodiazepines
Benzodiazepines are sedatives, and antiepileptic and antianxiety medications. The different ben-
zodiazepines vary in potency, duration of effect, and conversion to active and inactive metabo-
lites. Benzodiazepines produce a generalized depression of spinal reflexes and may cause coma.
Death from benzodiazepine overdose is rare unless the drugs are used in combination with other
compounds, such as alcohol. Oral overdoses of diazepam (Valium®) have been reported in excess
of 15 to 20 times the therapeutic dose without serious depression of consciousness. However,
if the same drug is given at a much lower concentration with rapid intravenous injection, respi-
ratory arrest can occur. Although there is variability among benzodiazepines, the onset of CNS
depression is typically observed 30 to 120 minutes after ingestion. Drug levels can be obtained
from both serum and urine specimens. However, because levels are rarely of value in emergency
management, quantitative analysis is often not conducted. Of the benzodiazepines, clonazepam,
used in the treatment of absence seizures, is the most often monitored, especially in children,
although most patients are managed without monitoring. There is little evidence for a therapeutic
window, probably because receptor effects do not mirror plasma concentrations, and tolerance
can develop with continued use. HPLC is used for quantitative analysis in serum/plasma, but the
presence of drug can be detected in urine by immunoassay.
Cannabinoids
Cannabis derivatives include marijuana and hashish. Marijuana consists of the leaves and flow-
ering parts of the plant Cannabis sativa. Marijuana is usually smoked in cigarettes or pipes, and
can be ingested in food. Dried resin from the plant can be compressed into blocks to make hash-
ish. The primary psychoactive cannabinoid in marijuana is delta-9-tetrahydrocannabinol (THC).
THC is also available in capsule form as a treatment for nausea in patients being treated with
chemotherapeutic agents and those undergoing treatment for glaucoma. The effects of THC are
related to the dose and time after consumption. THC may be a stimulant, a sedative, or a halluci-
A urine test for
nogenic compound. A typical marijuana cigarette contains 1% to 3% THC, but some may contain
cannabinoids may be
positive for 10 to 25 days
up to 15% THC. Hashish contains 3% to 6% THC, and an oil extract can be prepared from hash-
after the last exposure in ish with 30% to 50% THC. Significant variability in toxicity exists between individuals, which is
moderate to heavy use. influenced by prior exposure to the drug and degree of tolerance. The clinical presentation of a
In fact, there are well- patient after use of THC can vary from euphoria and a heightened sensory awareness to impaired
documented reports of short-term memory, depersonalization, visual hallucinations, and acute paranoid psychosis. THC
true-positive test results use can be established by detection of the drug in the urine. However, drug levels correlate poorly
in chronic use more with the degree of intoxication. A urine test for cannabinoids may be positive for 10 to 25 days
than 80 days after last after the last exposure in moderate to heavy use. In fact, there are well-documented reports of
exposure to THC. true-positive test results in chronic use more than 80 days after last exposure to THC.
Cocaine
Cocaine is a stimulant drug. It may be sniffed into the nose, smoked, or injected intravenously.
The “free base” form of cocaine is preferred for smoking because it volatilizes at a lower tempera-
ture and is not as easily destroyed by heat as the hydrochloride salt of the drug. Crack cocaine
is a dried form of the drug that has been mixed in alkaline aqueous solution to generate the
free base. Cocaine can also be combined with heroin and injected as a “speed ball.” The primary
effect of cocaine is generalized sympathetic stimulation, very similar to that produced by amphet-
amines. There is also a depression of cardiovascular function as a result of decreased cardiac
contractility. The toxic dose depends significantly on the tolerance of the individual to the drug,
the route of administration, and whether the cocaine is administered with other compounds.
A dose that produces only euphoria when swallowed or snorted can produce convulsions and
cardiac arrhythmias when rapidly injected intravenously or smoked. The initial euphoria from
exposure to cocaine can be followed by anxiety, agitation, hyperactivity, and seizures. With high
doses, respiratory arrest can occur. Death can result from fatal arrhythmia, status epilepticus,
intracranial hemorrhage, or hyperthermia. Cocaine use can be detected through analysis of the
primary metabolite, benzoylecgonine, in the urine, although parent drug and metabolites can
also be analyzed in plasma/serum or in vitreous humor in death investigations.
Opiates and Opioids

Opiates are narcotic sedatives and analgesics used for pain management. They are naturally
occurring compounds extracted from the poppy Papaver somniferum. Opioids include the natu-
rally occurring opiates and their derivatives (morphine and codeine) and the synthetic opioids
(dihydrocodeine, heroin, hydrocodone, hydromorphone, oxycodone, and oxymorphone). Many
prescription medications contain opioids. Mixtures of aspirin or acetaminophen with an opi-
oid compound, such as codeine, are in common use. Dextromethorphan is an opioid derivative
that is used to suppress cough. This compound can be obtained without a prescription as it has
no analgesic or addictive properties. Morphine is an opiate that is widely used in medicine to
reduce pain. The best known drug of abuse in this category is heroin. In general, all opiates and
opioids cause sedation and respiratory depression. Toxicity is related to respiratory failure that
can lead to death. The toxic dose varies widely with the opioid administered, its route and rate of
administration, and the individual’s tolerance to the drug. Diagnosis of opiate intoxication may
be established clinically when the typical signs and symptoms are present—pinpoint pupils, and
respiratory and CNS depression. These symptoms are reversed by administration of the opioid
antagonist naloxone. Opioid use can be determined in urine using immunoassay. Specific immu-
noassays can detect the presence of methadone, fentanyl, and buprenorphine while chromato-
graphic techniques can analyze meperidine and tramadol. Levels of these compounds in serum/
plasma, however, are not usually analyzed, because opiate concentration correlates poorly with
clinical effects.
Oxycodone is synthesized from thebane, a natural constituent of opium. It is available in a
number of compound analgesics, with acetaminophen and aspirin. Oxycodone abuse has grown
over the last decade, and specific immunoassays are available for this drug, since most broad-
spectrum opiate class immunoassays fail to detect oxycodone in the clinically relevant concentra-
tion range.
Phencyclidine
PCP is an anesthetic agent that became popular as an inexpensive street drug in the late 1960s.
It is most often smoked, but it can be snorted, ingested orally, or injected. It is commonly used in
combination with other illicit drugs. Ingestion of PCP produces a generalized loss of pain percep-
tion and can cause hallucinations, euphoria, and disinhibition. Ingestion of large amounts can
produce death, often from self-destructive behavior or from complications of hyperthermia. PCP
use can be detected in the urine using immunoassays. PCP levels in the serum are not clinically
valuable, because drug levels do not correlate with the degree of intoxication.
Alcohols: Ethanol, Methanol, Ethylene Glycol, and Isopropanol

Ethanol is the most common drug of abuse. Many patients presenting to hospitals with altered Ethanol is the most
mental status suffer from excess ethanol ingestion. Ethanol is present not only in beverages but common drug of abuse.
also in many medications. Ethanol intoxication is associated with many different types of acci- Many patients presenting
dental injury, particularly those involving motor vehicles. Chronic abuse of ethanol can lead to to hospitals with altered
pancreatic disease and liver cirrhosis. mental status suffer from
Ethanol is rapidly absorbed from the gastrointestinal tract. It distributes into the total body excess ethanol intake.
water and diffuses freely into the tissues. Peak blood ethanol levels occur 30 to 75 minutes after
ethanol ingestion. Food ingestion can delay absorption. A useful rule of thumb is that 1 oz of
80 to 100 proof spirits, 4 oz of wine, or 12 oz of beer increases the blood alcohol concentra-
tion by 25 to 30 mg/dL when ingested over a period of several minutes. The blood ethanol
level in a nonchronic alcoholic decreases at a rate of 15 to 25 mg/dL/h once ethanol ingestion
is discontinued. The blood ethanol levels required to induce fetal alcohol syndrome have not
been determined. Pregnant women who abuse ethanol have a high risk of delivering an infant
with fetal alcohol syndrome. These infants have prenatal growth retardation, dysfunction of
the CNS, and characteristic craniofacial abnormalities. Because an acceptable lower limit of
alcohol intake in pregnancy has not been defined, pregnant women are generally advised to
abstain from ethanol.
Metabolism of ethanol occurs by an oxidative pathway to acetaldehyde and acetic acid, and,
also, by a nonoxidative pathway to fatty acid ethyl esters. When the acetaldehyde is subsequently
converted to acetic acid, acidosis can occur. The major metabolizing enzyme for oxidation of
ethanol to acetaldehyde is alcohol dehydrogenase, and a second enzyme is the cytochrome
P450 system. The cytochrome P450 enzymes are liver microsomal enzymes involved in the
metabolism of several drugs. Cytochrome P450 enzymes can be induced to higher levels of
activity by ethanol. Ingestion of ethanol can thus alter the metabolism of a number of drugs,
which are metabolized by this same system. Induction may increase or decrease the therapeutic
and/or toxic effect of drugs. Ethanol can also compete with drugs for metabolism by the cyto-
chrome P450 enzymes.
The measurement The measurement of blood ethanol concentration can be performed by breath analysis, or
of blood ethanol more accurately, by a specific enzymatic assay or by gas chromatography that can also measure
concentration can be ethanol, methanol, and isopropanol, individually (Table 6–9). Methanol and isopropanol are
performed by breath also toxic, primarily as a result of oxidative metabolism. Methanol metabolism can generate
analysis, or more formic acid leading to blindness, while isopropanol is metabolized to acetone causing keto-
accurately, by an sis. Ethylene glycol, another volatile compound, is oxidized by similar metabolism to oxalic
enzymatic assay for acid causing acute kidney failure. Gas chromatography under different laboratory conditions
ethanol specifically or is required to detect ethylene glycol. The clinical presentation, sources for ingestion, and labo-
a gas chromatographic
ratory detection and quantitation of methanol, ethylene glycol, and isopropanol are shown in
test that can measure
Table 6–10.
ethanol, methanol, and
isopropanol, individually.
TABLE 6–9 Laboratory Evaluation for Ethanol Intake

Laboratory monitoring: acute intake Blood ethanol level (see below)
Laboratory monitoring: chronic intake Long-term markers of ethanol intake include elevated gamma-
glutamyl transferase (GGT), carbohydrate-deficient transferrin, and
fatty acid ethyl esters
Liver function tests AST (SGOT), ALT (SGPT), and bilirubin assess ethanol-induced liver
injury (see Chapter 16 for a discussion of cirrhosis)
Pancreatic function tests Amylase and lipase can be used to assess ethanol-induced pancreatic
injury (see Chapter 17 for a discussion of pancreatitis)
Blood Ethanol Concentration (mg/ Influence of Blood Alcohol Concentration in Individuals Who Are
dL) (Ranges Overlap Because of Not Chronic Ethanol Abusers
Person-to-person Variability)
10-50 Sobriety
40-120 Euphoria
90-250 Excitement
180-300 Confusion
270-400 Stupor
350-500 Coma
>450 Death can be produced by ingestion of 300-400 mL of pure ethanol

or 600-800 mL of 100 proof whiskey in <1 h
Data from Dubowski KM. Alcohol determination in the clinical laboratory. Am J Clin Pathol. 1980;74:747–750.
TABLE 6–10 Methanol, Ethylene Glycol, and Isopropanol Toxicity and Laboratory
Monitoring
Methanol Ethylene Glycol Isopropanol
Sources for ingestion Methanol, methanol- Antifreeze Rubbing alcohol

contaminated
alcohols, and
antifreeze
Time until onset of symptoms 12-48 h 0.5-12 h Minutes
Fatal dose of the pure 60-250 mL in most Approximately 100 g Approximately 250 mL
compound cases
Clinical features Impaired vision up to Anuria, vomiting, Vomiting, abdominal

blindness, vomiting, seizures, coma pain, hematemesis,
seizures, coma melena, coma
Antidote administration 4MP; ethanol 4MP; ethanol None (hemodialysis

>400 mg/dL)
Laboratory monitoring
Presence in blood Yes Yes Yes
Osmolality (mOsm = [mg/dL Elevated Elevated Elevated

alcohol]/[10 × molecular weight])
Hypoglycemia Yes Yes No

Acidosis (blood pH) Severe Severe Mild
Oxalate crystals (urine) No Yes (because ethylene No

glycol is metabolized
to oxalate)
Anion gap Large Large Normal

4MP, 4-methylpyrazole.
ENVIRONMENTAL TOXINS
Overview
Monitoring of environmental toxins is a significant challenge because so many substances that
can produce illness and even death are encountered in daily life. Occupational exposure to heavy
metals, gases, and caustic compounds can occur in the workplace, and leaching of toxins from
an industry can contaminate soil and groundwater, leading to exposure of food sources, drinking
water supplies, and livestock. Carbon monoxide, mercury, cyanide, and insecticides are some of
the notable environmental toxins. Lead exposure can occur from occupational and nonoccupa-
tional sources (such as paint chips) and produce subclinical to life-threatening illness, depending
on the amount ingested. Low-level exposure in children can produce serious disease and affect
long-term mental development.
Laboratory analysis of an environmental toxin may not always measure the compound Carbon monoxide
directly. In some cases, the toxin impairs flow through a metabolic pathway. Accumulated metab- poisoning is responsible
olites can be measured that reflect the toxic effects rather than a direct analysis of the toxin. Insec- for up to 4000 deaths per
ticide exposure, for instance, can be measured indirectly by cholinesterase enzyme activity rather year in the United States
than direct analysis of the insecticide levels in the body. and is the leading cause of
accidental and deliberate
poisonings. The principal
Carbon Monoxide pathologic consequence
of carbon monoxide
Description poisoning is the binding
Carbon monoxide poisoning is responsible for up to 4000 deaths per year in the United States of carbon monoxide to
and is the leading cause of accidental and deliberate poisonings. Approximately 10% of the cases oxygen-binding sites in
involve children. The heart, CNS, and lungs are the organs most immediately affected by the toxic the hemoglobin molecule.
TABLE 6–11 Clinical Presentation of Carbon Monoxide Toxicity

Carboxyhemoglobin Relative
to Total Hemoglobin (%) Clinical Findings in Adultsa
0.1-0.9 Normal range for nonsmoking adults
10-30 As concentration elevates, increasingly severe headache and greater

dyspnea on exertion
40-50 Very severe headache and dyspnea with tachycardia; may be fatal
60-70 Coma, seizures, often fatal
80 Rapidly fatal
a
Children are more sensitive and can present differently.
effects of carbon monoxide. Carbon monoxide can also impair vision, hearing, and peripheral
nerve conduction. The poisoning may be sublethal and cause cardiac dysrhythmias, myocardial
ischemia, headache, and a variety of other signs and symptoms. Survivors can suffer permanent,
severe neurologic impairment.
The principal pathologic consequence of carbon monoxide poisoning is the binding of car-
bon monoxide to oxygen-binding sites in the hemoglobin molecule. The binding of carbon mon-
oxide to hemoglobin results in the formation of carboxyhemoglobin. Carbon monoxide has a
higher affinity for hemoglobin than oxygen and decreases the hemoglobin’s ability to deliver oxy-
gen to the tissues producing ischemia.
The normal range for percent carboxyhemoglobin for adults is 0.1% to 0.9% of total hemo-
globin. When hemolytic disease is present with increased breakdown of hemoglobin, the
carboxyhemoglobin levels can increase to approximately 2%. Values at this level can have adverse
clinical effects in patients with preexisting heart disease. There is a poor correlation between
carboxyhemoglobin levels and clinical findings. Table 6–11 shows the relative consequences
of various amounts of carboxyhemoglobin, but individual patients demonstrate considerable
variability in clinical symptoms. Children are much more susceptible to acute carbon monoxide
poisoning and have a different clinical picture that mimics gastroenteritis. Like adults, they can
also have serious neurologic sequelae and myocardial ischemia. Patients are often unaware of
their exposure to carbon monoxide because it is odorless and nonirritating. There is no pathogno-
monic feature of carbon monoxide intoxication. A rapid diagnosis of carbon monoxide poisoning
is important in order to institute appropriate management and identify sources of carbon monox-
ide before other exposures can occur.
Diagnosis
Laboratory monitoring Laboratory monitoring of carbon monoxide poisoning is performed by measurement of the
of carbon monoxide carboxyhemoglobin levels (Table 6–12). The patient also must be evaluated for possible under-
poisoning is performed lying cause(s) of an increased carbon monoxide level (<10%), such as the presence of anemia,
by measurement of the infection, and smoking that can increase carboxyhemoglobin levels. Because carbon monoxide
carboxyhemoglobin
levels.
TABLE 6–12 Laboratory Tests Used in the Evaluation of a Patient for Carbon
Monoxide Poisoning
Laboratory Test Comments
Carboxyhemoglobin (% relative See Table 6–11

to total hemoglobin)
CBC/relevant microbiology Anemias and infections can increase the concentration of

studies carboxyhemoglobin and should be identified if present
Indicators of ischemic damage Creatinine kinase, troponin I, troponin T, lactate dehydrogenase, and/or
to skeletal and cardiac muscle aldolase may be elevated; myoglobin may be detectable in the urine if there
is muscle damage
can cause ischemic damage to skeletal and cardiac muscle, evaluation of ischemic muscle dam-
age may also be appropriate.
Lead
Description
Lead poisoning is primarily a disease of childhood. As research has increased about lead tox- As understanding
icity, the threshold for defining lead poisoning has decreased over the past 20 years. Prior to has increased about
1970, lead poisoning (plumbism) was defined by blood levels greater than 60 μg/dL. In 1971, the the toxicity of lead,
threshold was lowered to 40 μg/dL. By 1975, the acceptable level was 25 μg/dL and since 1985, the threshold for the
blood levels of 10 to 15 μg/dL have been recognized to impair cognitive and behavioral develop- definition of lead
ment. Most recently, the Centers for Disease Control and Prevention have adopted recommended poisoning has decreased
lead levels in children of <5 μg/dL. As recently as 1992, 17.2% of children in the United States over the past 20 years.
between the ages of 6 months and 5 years were estimated to have a blood lead level in excess of
15 μg/dL, although this pales in comparison to those observed in developing countries. These
children were primarily from low-income families in large urban settings. The sources of lead for
these children included not only lead paint but also lead-contaminated household dust, soil, and
workplace clothing; the use of lead-containing cookware; exposure to lead in storage batteries,
in fishing and curtain weights; and even lead-contaminated water from the use of lead-soldered
pipes in older buildings. Some canned food has been reported to contain lead. Lead-containing
costume jewelry, medicines, and cosmetics (such as surma or kohl) imported to the United States
from other countries have been demonstrated to contain lead. Obviously, the investigation of lead
poisoning cases is important to identify the precise source of lead ingestion so that exposure can
be eliminated.
There is a significant effort nationally to screen children, particularly those between the ages
of 6 months and 5 years who live in, or are frequent visitors to, deteriorated housing built prior
to 1960. Exposure to lead at high doses can produce persistent seizures, mental retardation, and
chronic behavioral dysfunction. Most of the absorbed lead is stored in the bones. However, lead
can also be found in soft tissues and erythrocytes. Lead interferes with the enzymes involved in
heme synthesis, so lead exposure can lead to anemia. Renal toxicity is also observed in some cases
of chronic lead poisoning. Any child with developmental delay, behavioral disorders, seizures,
learning disabilities, iron deficiency, hearing impairment, renal disorders, and recurrent vomiting
and abdominal pain should be considered for lead toxicity.
Diagnosis
A whole blood lead level reflects the lead burden of the body. For small children, a finger- A whole blood lead
stick sample is usually sent for analysis. Table 6–13 describes the laboratory monitoring for level reflects the lead
children suspected of lead poisoning relative to the presenting blood lead level. If the blood burden of the body.
lead level is greater than 5 μg/dL, testing of a sample taken by venipuncture is recommended For small children, a
to rule out skin contamination. If this “clean” specimen contains more than 5 μg/dL lead, finger-stick sample is
parental education on possible exposure sources is recommended. While initial testing is often usually sent for analysis.
performed by anodic stripping voltammetry, confirmation testing especially for concentra- If the blood lead level
tions above 40 μg/dL is generally performed by atomic absorption or mass spectrometry. Free is greater than 5 μg/dL,
erythrocyte protoporphyrin (zinc protoporphyrin) is formed from heme synthesis as a result testing of a sample taken
by venipuncture is
of lead toxicity. The measurement of free erythrocyte protoporphyrin is, however, an insensi-
recommended to rule out
tive screening test for lead exposure because it does not detect lead poisoning in children with
skin contamination.
lead levels between 10 and 25 μg/dL, and identifies less than 50% of children with blood levels
greater than 25 μg/dL. The utility of free protoporphyrin measurement is primarily to detect
ongoing lead exposure. Because the anemia from lead poisoning may resemble iron deficiency
anemia, studies for iron deficiency (such as serum ferritin and the red cell distribution width
in the complete blood count) should be obtained to differentiate anemia of lead poisoning
from iron deficiency anemia.
A portion of this chapter on TDM is also found in a newsletter to the physicians at the Massachusetts
General Hospital in Clinical Laboratory Reviews, 1999;8:1.
TABLE 6–13 Laboratory Monitoring of Children for Lead Poisoning (http://www.cdc.gov/nceh/lead/

casemanagement/caseManage_chap3.htm, Summary of Recommendations for Children With
Confirmed [Venous] Elevated Blood Lead Levels)
Blood Lead Level (μg/dL)
<5 5-44 45-69 ≥70
Lead education Lead education Lead education Hospitalize and commence chelation
• Dietary • Dietary • Dietary therapy (following confirmatory
• Environmental • Environmental • Environmental venous blood lead test) in
conjunction with consultation from
a medical toxicologist or a pediatric
environmental health specialty unit
Lead risk assessment and environmental Follow-up blood lead Follow-up blood lead Proceed according to actions for
sampling if appropriate monitoring monitoring 45-69 μg/dL
Complete history and Laboratory studies:
physical exam • Hemoglobin or hematocrit
• Iron status
Laboratory studies: • Free erythrocyte
• Iron status protoporphyrin
• Consider hemoglobin or
hematocrit Environmental investigation
Environmental investigation Lead hazard reduction
Lead hazard reduction Neurodevelopmental
monitoring
Neurodevelopmental
monitoring Abdominal x-ray (if particulate
lead ingestion is suspected)
Abdominal x-ray (if with bowel decontamination if
particulate lead ingestion indicated
is suspected) with bowel
decontamination if Oral chelation therapy.
indicated Consider hospitalization if
lead-safe environment cannot
be assured
The following actions are not recommended at any blood lead level: searching for gingival lead lines; testing of neurophysiologic function; evaluation of renal function (except
during chelation with EDTA); testing of hair, teeth, or fingernails for lead; radiographic imaging of long bones; x-ray fluorescence of long bones. For adults, it is recognized that
accumulation of lead occurs, and blood lead concentrations <25 μg/dL do not require action.
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C H A P T E R
Diseases of Infancy
and Childhood
Paul Steele 7
LEARNING OBJECTIVES
1. Identify the clinical testing of situations that indicate the need for prenatal
testing of mother and/or infant, and the clinical consequences of premature
birth.
2. Understand the rationale for selection of laboratory tests in neonatal
screening programs.
3. Learn the assessment for diagnosis of Down syndrome and the clinical
situations in which it is most often performed.
4. Learn the underlying defects that produce hemolytic disease of the newborn
and cystic fibrosis and the laboratory test abnormalities associated with these
disorders.
5. Learn the names of the diseases and the associated biochemical defects for
the more commonly encountered or better characterized inborn errors of
metabolism in the following categories:
• Amino acidurias not involving urea cycle enzymes
• Amino acidurias involving urea cycle enzymes
• Lysosomal storage diseases with impaired degradation of sphingolipids
• Lysosomal storage diseases with impaired degradation of
mucopolysaccharides
• Lysosomal storage diseases with impaired degradation of glycogen
CHAPTER OUTLINE
Introduction 186 Infectious Diseases in the Perinatal
Prenatal and Neonatal Laboratory Period 189
Testing 186 Hemolytic Disease of the Newborn 190
Prenatal Testing and Screening 186 Cystic Fibrosis 191
Neonatal Screening 186 Amino Acidurias 192
Neonatal Testing 186 Lysosomal Storage Diseases 193
Prematurity 187 Neuroblastoma 194
Down Syndrome 188
185
186 CHAPTER 7 Diseases of Infancy and Childhood
INTRODUCTION
It is difficult to precisely identify the diseases of infancy and childhood because many disorders
that begin in childhood become clinically evident in adulthood if a long period of time is required
to generate a pathologic lesion. The topics chosen for inclusion in this chapter are disorders pre-
senting almost exclusively in childhood. However, they obviously represent only a small fraction
of “childhood disorders.” Many disorders in other sections of this book, such as hemophilia and
numerous infections, occur or are diagnosed primarily in childhood. The chapter begins with an
overview of prenatal and neonatal laboratory testing.
PRENATAL AND NEONATAL LABORATORY TESTING

Prenatal Testing and Screening
The disorders that can be diagnosed before birth number in the thousands. In families in which
there is a history of a particular disorder, it is not uncommon to test prenatally for that particu-
lar disorder, often with DNA-based diagnostic tests. However, for the vast majority of families
without a history of a specific illness, prenatal screening may also be undertaken. A screen differs
from a test in that it does not provide a definitive diagnosis but rather an assessment of the risk
of a diagnosis. For most of these families, screening is preferred as an initial step because it is
less invasive; for example, there are several maternal serum screening assays (see below) for fetal
Down syndrome (also known as trisomy 21), but testing for fetal Down syndrome requires an
invasive procedure such as chorionic villus sampling or collection of amniotic fluid. The decision
to screen is a personal one for families and includes considerations such as parental age and desire
to avoid having a diseased child.
Neonatal screening was Neonatal Screening

introduced as a means to Neonatal screening was originally developed to detect diseases such as phenylketonuria (PKU)
detect disorders in which and congenital hypothyroidism, for which early detection and intervention could prevent cat-
immediate treatment can astrophic consequences such as intellectual disability (mental retardation). Improvements in
result in the prevention assay methodology, such as decreases in cost and the availability of tandem mass spectrom-
of catastrophic
etry for single assay detection of many abnormalities, have expanded the neonatal screening
consequences.
menu to include assays for diseases that are not preventable but are often treatable. All 50 US
states screen for over 26 disorders, including amino acidurias (such as PKU and maple syrup
urine disease), organic acidemias (such as isovaleric acidemia), fatty acid disorders (such as
medium-chain acyl-CoA dehydrogenase [MCAD] deficiency), hemoglobinopathies associated
with hemoglobin S, congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibro-
sis, and classical galactosemia, among others. As new methods and new assays are developed,
some variation between states’ neonatal screening menus will inevitably continue to exist; these
variations are tracked on a Web site maintained by the National Newborn Screening & Global
Resource Center.
As with any screening program, positive results require follow-up confirmation testing;
false-positives do occur. Suggested actions and algorithms for neonatal screen positives are
published on the Web by the American College of Medical Genetics and Genomics (ACMG).
Urgent intervention is required for some of these disorders, to preserve life or prevent intellectual
disability.
Neonatal Testing
The laboratory evaluation of an infant who appears clinically well in the first 24 hours of life but
develops signs of illness on the second or third day may include:
• Blood gases to detect metabolic acidosis/alkalosis
• Urinalysis to detect ketonuria
• Complete blood count to detect abnormalities in blood cells
• A blood glucose test to detect hypoglycemia
CHAPTER 7 Diseases of Infancy and Childhood 187
TABLE 7–1 Routine Laboratory Screening for Inherited Metabolic Disease

Laboratory Test Specimen
Lactate Blood, CSF if indicated
Pyruvate Blood
Amino acids Urine, blood, CSF if indicated
Organic acids Urine
Reducing sugars Urine
Glucose Blood, urine, CSF if indicated

Ketones and pH Urine
Liver enzymes, electrolytes, uric acid, ammonia Blood
Acylcarnitine profile Blood
Mucopolysaccharide screen Urine

CSF, cerebrospinal fluid.
• A blood ammonia test to detect elevated ammonia

• Liver function tests to detect hepatic dysfunction
• Prothrombin time and partial thromboplastin time to detect coagulopathies
• Blood lactate to detect lactic acidosis
Table 7–1 lists a number of screening laboratory tests that are typically ordered when there is
suspicion that a neonate (or older child) is suffering from an inborn error of metabolism.
The results of these tests only suggest specific disorders, with additional testing required to
identify a specific metabolic defect. Definitive tests to make a conclusive diagnosis of a metabolic
disorder often involve the measurement of specific enzyme activities or various metabolites in a
pathway. Because sepsis is often suspected, it must be ruled out in the sick infant if there are any A major cause of neonatal
signs or symptoms of infection. mortality and morbidity
is preterm labor and
delivery.
PREMATURITY
Description
A major cause of neonatal mortality and morbidity is prematurity, defined as birth prior to 37
weeks of gestation. When preterm labor or premature rupture of membranes causes prematurity,
the underlying etiology is not often apparent, although it is believed to be commonly associ-
ated with infection or inflammation. Maternal correlates of prematurity include diabetes, obesity,
intervention for infertility, genital or urinary infection, periodontal disease, low socioeconomic
status, and other factors. Conflicting information exists about the value of intervention such as
use of antibiotics for infection or infection risk.
Another cause of prematurity is iatrogenic, when the medical condition of the mother and/or
fetus compels intervention to produce early delivery. The timing of such elective intervention for
early delivery is influenced by the risk for fetal organ immaturity. Principal among these concerns
is lung immaturity that is associated with the development of respiratory distress syndrome in
the newborn.
Diagnosis
Risk of preterm delivery can be assessed by measurement of fetal fibronectin in cervical or vaginal
fluid. This glycoprotein is produced by fetal membranes and appears in the cervix and vagina early
in pregnancy as implantation develops, but normally disappears by week 20. Its reappearance in
the third trimester often precedes labor and delivery. Its chief clinical value lies in its negative
predictive value, that is, patients thought to be at risk for preterm labor who are negative for fetal
fibronectin in their cervicovaginal fluid are very unlikely to deliver within 1 week of the laboratory
result. The major barrier to the widespread use of the fetal fibronectin test, when positive, is that
clinical interventions to end preterm labor are only partially successful.
In those instances when fetal or maternal health dictates early delivery, there are several tests
available to assess fetal lung maturity. A simple and inexpensive test is to count lamellar bodies
in amniotic fluid, using the platelet channel in a conventional hematology automated analyzer.
These lamellar bodies are surfactant-containing products of Type II pneumocytes. The finding
of greater than 50,000 lamellar bodies per microliter of amniotic fluid predicts lung maturity. If
fewer bodies are present, further testing on the amniotic fluid sample is warranted. Other tests
include identification of the presence of phosphatidylglycerol (PG), and determination of the
ratio of lecithin to sphingomyelin (L/S ratio). (See Chapter 14 for additional information.)
DOWN SYNDROME
Description
Down syndrome is the most commonly encountered, clinically significant autosomal chromosome
aberration affecting individuals beyond infancy. This genetic defect, which can be detected by
cytogenetic analysis, is trisomy 21. More than 90% of Down cases occur as a result of meiotic
Down syndrome is nondisjunction. Down syndrome is characterized by intellectual disability, cardiac malformations,
the most commonly malformations of the digestive tract, eyes, and ears, and the development of an Alzheimer-like
encountered, clinically disease process in later life.
significant autosomal The overall birth prevalence of Down syndrome is approximately 1 in 1000 births. However,
chromosome aberration a woman’s individual risk to deliver an infant with Down syndrome depends substantially on her
affecting individuals age. The risk increases significantly past age 35 years, with an incidence in the range of 1:270 to
beyond infancy. This 1:100 by age 40 years.
genetic defect, which
can be detected by
cytogenetic analysis, is Screening and Diagnosis
trisomy 21. The neonatal The neonatal diagnosis of Down syndrome is clinical, with metaphase chromosome analysis on
diagnosis of Down peripheral blood serving merely to confirm the diagnosis.
syndrome is clinical, with Noninvasive fetal screening for Down syndrome involves many more tests (Table 7–2) that
metaphase chromosome are used in combination to develop a risk assessment of Down syndrome during pregnancy.
analysis on peripheral Definitive diagnosis of fetal Down syndrome during pregnancy is established by an invasive test,
blood serving merely to namely, metaphase analysis of cells from either chorionic villus sampling (typically limited to
confirm the diagnosis. first trimester) or amniotic fluid collection. The decision to engage in fetal screening for Down
syndrome or to move from screening tests to invasive diagnostic testing once a risk assessment is
completed depends on patient preference. The invasive tests to assess for Down syndrome during
pregnancy do carry a risk of miscarriage.
First-trimester screening typically consists of measurement of 2 analytes in maternal serum:
pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic
gonadotropin (fβhCG); the former is low and the latter high in mothers carrying a Down syn-
drome fetus. A third part of first-trimester screening is ultrasound assessment for nuchal trans-
lucency, which is increased as a result of fluid accumulation in the neck of a Down syndrome
fetus. This first-trimester screening is associated with a sensitivity of approximately 85%, with a
5% false-positive rate. Nuchal translucency alone is not recommended in singleton pregnancy
because its sensitivity is only about 70%. However, in multiple gestation pregnancies, the interpre-
tation of maternal serum markers can be problematic, while the nuchal translucency test permits
evaluation of each fetus. Determination of nuchal translucency is highly operator-dependent and
requires specific training.
Second-trimester screening typically consists of the so-called quadruple screen of mater-
nal serum, consisting of measurement of the following analytes: alpha-fetoprotein, unconjugated
estriol (both decreased in mothers carrying a Down syndrome fetus), and total hCG and inhibin
A (both increased in such mothers). The quadruple screen has a detection rate of approximately
81% with a 5% false-positive rate. An older test, the “triple screen” includes all of these second-
trimester markers except inhibin A. It is characterized by higher false-positive rates and lower
TABLE 7–2 Laboratory Evaluation for Down Syndrome

First-trimester screen
Pregnancy-associated plasma protein A (PAPP-A) Low in pregnancy with Down syndrome fetus
Free beta hCG Elevated in pregnancy with Down syndrome fetus
Nuchal translucency Ultrasound exam; permits evaluation of each fetus in

multiple gestation pregnancy
Quadruple screen Second-trimester screen
Alpha-fetoprotein (AFP) Low in pregnancy with Down syndrome fetus; elevated

with fetal neural tube defect
hCG Elevated in pregnancy with Down syndrome fetus
Unconjugated estriol (UE3) Low in pregnancy with Down syndrome fetus
Inhibin A Elevated in pregnancy with Down syndrome fetus
DNA sequence of circulating cell-free fetal DNA Can detect trisomy 21 as well as other defects including
trisomy 18 and trisomy 13
Metaphase chromosome analysis Diagnostic test; can be performed on chorionic villus

sample, cells from amniotic fluid, and newborn blood
detection rates. Maternal serum results are typically described in the form of “multiples of the
median” (or MoM); the normal range is highly dependent on several factors including gestational
age, number of gestations, maternal weight, and race.
Combining first- and second-trimester screens can provide an even higher level of
detection. One approach is to sequentially conduct the tests; that is, inform the patient of the
results of the first-trimester screen as soon as they are available (this permits her to choose a
more definitive diagnostic method if indicated), and later perform the quadruple screen in the
second trimester if appropriate. A noninvasive test that rivals the sensitivity and specificity of
invasive testing is DNA sequencing of circulating cell-free fetal DNA (ccffDNA) in maternal
serum. This test could also identify other chromosome aneuploidy syndromes such as trisomy
18 and trisomy 13. Current recommendations for follow-up of positive ccffDNA sequencing
results are to confirm the abnormality by invasive testing (metaphase analysis on chorionic
villus or amniotic fluid samples), but a negative ccffDNA sequencing test in a patient whose
first- or second-trimester screen is positive for Down syndrome may provide an option to forgo
invasive testing.
A final point about maternal serum screening is that the alpha-fetoprotein assay in the qua-
druple screen, if elevated, provides a measure of increased risk for neural tube defects such as
spina bifida. These cases can be further studied by amniotic fluid collection, with assessment A number of maternal
of acetylcholinesterase as well as alpha-fetoprotein (both elevated with neural tube defects) and infections affect the
high-resolution ultrasound examination. fetus and newborn.
Neonatal death has been
linked to a number of
INFECTIOUS DISEASES IN THE PERINATAL PERIOD infections, including
cytomegalovirus (CMV),
Description group B streptococcus,
A number of maternal infections affect the fetus and newborn. Bacterial vaginosis, sexually herpes simplex, Listeria,
transmitted diseases, and others increase the risk of preterm labor. Rubella and syphilis are parvovirus, and others.
associated with congenital anomalies. Neonatal death has been linked to a number of infec-
tions, including cytomegalovirus (CMV), group B streptococcus, herpes simplex, Listeria, par-
vovirus, among others. Postnatal disease in the offspring occurs with many infections, such as
hepatitis B and C, human immunodeficiency disease (HIV), CMV, rubella, toxoplasmosis, and
syphilis.
Screening and Diagnosis

Routine prenatal care is designed to screen for several of these infections, for the purpose of iden-
tifying pregnant women who need intervention or identifying susceptibility for a poor outcome in
the mother. The results of maternal screening tests may have implications for the fetus, especially
when they indicate maternal infection during pregnancy. An example of the latter is rubella serol-
ogy screening of maternal serum. Routine testing includes serologic testing for syphilis, hepatitis B
and C, and HIV. Routine rectovaginal culture for group B streptococcus is performed late in the
third trimester. Detection through nucleic acid testing and/or culture is carried out for Chlamydia
and for gonorrhea in high-risk mothers, and for herpes simplex in mothers with genital lesions.
HEMOLYTIC DISEASE OF THE NEWBORN

Description
Hemolytic disease of the newborn (HDN), also known as erythroblastosis fetalis, is a syndrome
in which the newborn becomes anemic from the destruction of his/her RBCs in utero. This RBC
destruction is a result of maternal IgG antibodies formed against a red cell antigen, most com-
monly the Rh antigen (also known as D antigen), which are then delivered into the fetal circu-
lation across the placenta. Antibody production by a mother who is Rh-negative results from
exposure to Rh-positive fetal cells during pregnancy and, to a much greater extent, at delivery.
Hemolytic disease of the Therefore, the women at greatest risk for delivering infants with HDN are Rh-negative mothers
newborn (HDN), also who conceive Rh-positive babies, and are in the second or subsequent pregnancies. It is general
known as erythroblastosis practice to identify risk of sensitization during pregnancy and treat the mother prophylactically
fetalis, is a syndrome by rapidly removing Rh-positive fetal cells through passive immunization with Rh immune glob-
in which the newborn ulin. Such immunizations are almost always effective in preventing the mother’s immune system
becomes anemic from from developing these alloantibodies. Treatment is employed not only following delivery, when
the destruction of his/her fetal to maternal bleeding is expected, but also during pregnancy itself.
RBCs in utero. Other red cell alloantibodies may be involved, although much less commonly. A form of
HDN, usually mild, results from transplacental passage of IgG-class antibodies against A or B
red cell antigens, to a Type A, B, or AB fetus. This disorder may occur with the first pregnancy, as
it involves maternal antibodies that normally arise without the requirement of a previous preg-
nancy or incompatible blood transfusion.
Neonatal disease related to maternal antibody may occasionally involve targets other than
RBC antigens; examples include neonatal alloimmune thrombocytopenic purpura (NAIT) with
maternal antiplatelet antibodies.

ABO/Rh typing is used in routine prenatal care to identify the mother’s blood type. An anti-
body screen against a standard panel of red cells, employing the indirect antiglobulin method
(see Chapter 2), is also routinely performed to determine if there are maternal alloantibodies
(such as anti-Rh) that might be a threat to the fetus.
Testing for fetal blood type (which establishes presence or absence of fetal susceptibility
in that pregnancy), as well as monitoring for fetal anemia and hyperbilirubinemia (the
latter a consequence of the RBC destruction), typically requires invasive procedures such as
amniocentesis (withdrawal of amniotic fluid) or cordocentesis (withdrawal of blood from
the cord, in utero). Both of these carry some risk of pregnancy loss or fetal damage. A new,
noninvasive approach is possible with genetic testing for the antigen genes. Genotyping
of the father that reveals homozygosity of the antigen gene implies fetal antigen positivity.
Heterozygosity in the father implies a 50% chance of fetal antigen negativity, in which
case there would be absence of risk for HDN. Confirmation of fetal antigen negativity can
be accomplished by genotyping of fetal DNA that is present in maternal plasma. Titering the
quantity of maternal antibody as a disease predictor in the fetus has been used, but the amount
of antibody does not always correlate with the severity of the disease. Testing of amniotic fluid
for bilirubin by spectrophotometric analysis can be performed to help manage the disease;
fetuses at high risk of severe anemia, based on the amniotic fluid bilirubin, would be delivered
if tests of fetal lung maturity on the amniotic fluid sample (see above) reveal a low risk of
TABLE 7–3 Laboratory Evaluation of the Mother and Newborn for Hemolytic
Disease of the Newborn (HDN)
Result/Comment
To predict HDN during pregnancy
Maternal ABO and Rh type Provides screening for possible HDN risk
Maternal antibody screen Detects many common anti-red cell antibodies
Maternal antibody titer Significant elevation implies risk of HDN

Genotyping of father Homozygosity for antigen gene implies fetal risk; heterozygosity for antigen
gene requires further testing to assess fetal susceptibility
Genotyping of fetus Presence of antigen gene can be assessed; specimen may be amniotic fluid
or cord cells (both invasive) or maternal plasma
To assess for HDN during pregnancy
ΔOD450 Invasive; spectrophotometric assessment of bilirubin through assessment

of change (delta) of optical density at 450 nm from expected to observed
Fetal hematocrit Invasive; requires cordocentesis
Fetal middle cerebral Noninvasive; peak velocity of systolic blood flow increases progressively
artery flow with fetal anemia
To evaluate disease in the newborn

Clinical findings Infant may appear normal to very abnormal; jaundice is common;
cardiorespiratory problems may occur; severely affected fetuses may die
in utero or at delivery
Reticulocyte count At 7 days of life, an elevated level is consistent with HDN
Nucleated red blood cells A persistently high percentage of nucleated red blood cells is consistent
with increased red blood cell production
Unconjugated bilirubin Markedly elevated, but other entities, such as liver immaturity, can cause
an increased unconjugated bilirubin in neonates
Haptoglobin Low value indicates intravascular hemolysis
Direct antiglobulin test Positive result indicates maternal antibodies to red blood cells in the
newborn circulation
respiratory distress syndrome. Otherwise, intrauterine blood transfusion and exchange could
be performed. A noninvasive ultrasound test can detect fetal middle cerebral artery blood flow
rates that correlate well with the presence of fetal anemia. This test has been shown to be more
sensitive, specific, and accurate than the invasive amniotic fluid bilirubin measurement.
Laboratory evaluation of newborns for HDN includes complete blood count and direct anti-
globulin test (DAT) on cord blood. The latter test detects the presence of immunoglobulin and/
or complement deposited on the surface of red blood cells. See Table 7–3 for a list of tests that are
helpful in the laboratory evaluation of HDN.
CYSTIC FIBROSIS Cystic fibrosis is an

autosomal recessive
Description disease that results from
Cystic fibrosis is an autosomal recessive disease that results from a mutation in the cystic fibrosis a mutation in the cystic
transmembrane conductance regulator (CFTR) gene on the long arm of chromosome 7. The clini- fibrosis transmembrane
cal presentation is dysfunction of exocrine glands, from abnormal chloride conduction across the conductance regulator
apical membrane of epithelial cells, and subsequently chronic obstructive lung disease and exo- (CFTR) gene on the long
crine pancreatic insufficiency. The most commonly found mutation in the CFTR gene is ΔF508 arm of chromosome 7.
(loss of a phenylalanine codon at position 508), and it is present in approximately 70% of cases.
However, more than 1800 mutations in this gene have been described, most of which are rare and
some of which are equivocally associated with disease.
TABLE 7–4 Laboratory Evaluation for Cystic Fibrosis

Quantitative pilocarpine Sweat chloride concentration >60 mEq/L confirms the diagnosis,
iontophoresis sweat chloride test in the presence of supporting clinical and laboratory information;
differential diagnosis for a positive sweat test result includes untreated
adrenal insufficiency, hereditary nephrogenic diabetes insipidus,
hypothyroidism, pancreatitis, and malnutrition
Genetic testing Used for carrier testing, disease testing, and newborn screen follow-up
testing
72-h fecal fat level Elevated
Fecal elastase Decreased
Immunoreactive trypsinogen Elevated in newborn screening test
The incidence of cystic fibrosis is approximately 1 in 2500 to 3400 Caucasian births, 1 in

17,000 births from individuals of African descent, and 1 in 90,000 Asian births. As many as 1 in
29 Caucasians may be a carrier for cystic fibrosis.

Extending an offer to screen the carrier status of couples planning for pregnancy is now recom-
mended, especially for Caucasians (who are at highest risk) and those with a family history of
disease. The carrier screen is a molecular genetic test designed to detect the most common muta-
tions, typically around 23 in number.
Screening newborns for the disease is increasingly common. The usual approach is to screen
blood spots for immunoreactive trypsinogen (IRT) and then follow up elevated values with either
a multiple-mutation DNA test or repeat IRT on a new sample.
Laboratory evaluation for screen-positive patients includes the same test used in the assess-
ment of patients clinically suspected of having the disease: quantitative sweat chloride. This analy-
sis involves testing to determine whether the patient exhibits elevated levels of chloride in sweat
samples; these samples are typically elicited with the use of pilocarpine and iontophoresis.
Genetic testing for disease detection (as opposed to screening) involves a larger number of
mutation investigations, typically more than 75. If necessary, nucleotide sequencing of the gene
can be performed; this procedure would typically be limited to the proband within a family; if an
unusual mutation is found, other family members can be tested with sequencing of DNA from the
involved exon rather than the entire gene.
Five classes of gene mutations, each specific for a different type of CFTR malfunction, have
been described. The development of CFTR-modulating drugs, specific for a particular class of
mutations, makes the genetic characterization of CF all the more important. Table 7–4 presents
the laboratory evaluation of cystic fibrosis.
Amino acidurias are

AMINO ACIDURIAS
defects in the metabolism Description and Diagnosis
of amino acids that lead
Amino acidurias are defects in the metabolism of amino acids that lead to their accumulation.
to their accumulation. The
primary amino acidurias
The primary amino acidurias are a result of an inherited enzyme defect within a degradative
are a result of an inherited pathway for a specific amino acid, or in a transporter in the renal tubules, that alters the
enzyme defect within a reabsorption of the amino acid. Selected primary amino acidurias associated with impaired
degradative pathway for amino acid degradation are shown in Table 7–5. The defective enzymes and the amino acid or
a specific amino acid, or other compound in elevated concentration are listed for the individual disorders. In contrast
in a transporter in the to primary amino acidurias, secondary amino acidurias are accumulations of amino acids that
renal tubules, that alters arise when the organs responsible for their elimination or degradation, notably the liver and
the reabsorption of the kidney, are functionally impaired. The diagnosis of a primary amino aciduria may be made by
amino acid. demonstrating a decrease in a specific enzyme activity required for the metabolism of the amino
TABLE 7–5 Selected Amino Acidurias

Amino Acid or Other Compound in Elevated
Disorder Defective Enzymes Concentration (Most Prominent Ones)
For enzymes outside the urea cycle
Phenylketonuria (multiple forms of Phenylalanine hydroxylase Phenylalanine

the disorder with different enzyme Dihydropteridine reductase
deficiencies) Defect in biopterin synthesis
Tyrosinemia (multiple forms of the disorder Fumarylacetoacetate hydrolase Tyrosine

with different enzyme deficiencies) Tyrosine aminotransferase
Alkaptonuria Homogentisic acid oxidase Homogentisic acid

Homocystinuria (multiple forms of Cystathionine β-synthase Homocysteine
the disorder with different enzyme Methylenetetrahydrofolate reductase
deficiencies) (MTHFR)
Methyltransferase
Histidinemia Histidase Histidine
Maple syrup urine disease Branched-chain ketoacid decarboxylase Leucine, isoleucine, alloisoleucine, valine, and
corresponding ketoacids (in acute attacks)
Nonketotic hyperglycemia Block in glycine cleavage enzyme system Glycine
Methylmalonic acidemia Methylmalonyl-CoA mutase Glycine and methylmalonic acid

Cystathioninuria γ-Cystathionase Cystathionine
Carnosinemia Carnosinase Carnosine
Hyperprolinemia (multiple forms of Proline oxidase Proline

the disorder with different enzyme Δ5-Pyrroline-5-carboxylic acid
deficiencies) dehydrogenase
For enzymes within the urea cycle
Citrullinemia Argininosuccinate synthetase Citrulline, ammonia, and alanine
Argininosuccinic aciduria Argininosuccinate lyase Argininosuccinic acid, and citrulline; ammonia after meals
Argininemia Arginase Arginine; ammonia after meals
Hyperornithinemia Ornithine decarboxylase Ornithine, glutamine, and alanine; ammonia after meals
Ornithine transcarbamylase deficiency Ornithine transcarbamylase Ammonia and glutamine

Carbamoylphosphate synthetase deficiency Carbamoylphosphate synthetase Ammonia, glycine, and glutamine
acid or by finding the associated abnormality in the gene coding for the enzyme. The presence
of characteristic clinical signs and symptoms for the individual disorders is highly contributory
toward establishing a diagnosis. The different amino acidurias vary from essentially benign, with
no apparent disease, to lethal disorders. Furthermore, the clinical features can vary widely within
a single disease, because some of the amino acidurias have multiple forms and because some
enzyme deficiencies can result in an elevation of the same amino acid.
The lysosomal storage
diseases result from the
LYSOSOMAL STORAGE DISEASES lysosomal accumulation
of compounds that should
otherwise be degraded
Lysosomal storage diseases, like the amino acidurias, are inborn errors of metabolism. The lyso- by the enzymes in the
somal storage diseases result from the lysosomal accumulation of compounds that should other- lysosome. The diagnosis
wise be degraded by the enzymes in the lysosome. The diagnosis of a lysosomal storage disease of a lysosomal storage
is made by identifying the products stored in the tissues or excreted in the urine. Demonstration disease is made by
of an enzyme deficiency is usually sufficient for diagnosis of a specific lysosomal storage disease. identifying the products
With some disorders, DNA analysis for the mutation producing the enzyme deficiency also can be stored in the tissues or
performed. As with the amino acidurias, a number of lysosomal storage disorders have subtypes, excreted in the urine.
TABLE 7–6 Selected Lysosomal Storage Disorders

The Compound Stored in the Lysosome
Enzyme Deficiency
and Associated Disease
Sphingolipidosesa
Niemann–Pick disease Sphingomyelinase
Gaucher disease β-Glucosidase
Krabbe disease Galactosylceramide β-galactosidase
Metachromatic leukodystrophy Arylsulfatase A
Fabry disease α-Galactosidase (X-linked)
Tay–Sachs disease β-N-Acetylglucosaminidase A
Sandhoff disease β-N-Acetylglucosaminidase A and B
Generalized gangliosidosis β-Galactosidase
Mucopolysaccharidoses (MPS) b
Hurler syndrome α-L-Iduronidase

Scheie syndrome α-L-Iduronidase
Hunter syndrome Iduronate sulfatases (X-linked)
Sanfilippo syndrome (multiple forms of the disorder Heparan N-sulfatase
with different enzyme deficiencies) α-N-Acetylglucosaminidase
α-Glucosaminide-N-acetyltransferase
N-Acetylglucosamine-6-sulfate sulfatase
Morquio disease (multiple forms with different Galactosamine-6-sulfate sulfatase
enzyme deficiencies) β-Galactosidase
Maroteaux–Lamy disease Arylsulfatase B
Glucuronidase deficiency β-Glucuronidase
Glycogen storage disorders c
Pompe disease α-Glucosidase

a
Accumulation of sphingolipids (which includes sphingomyelins, glucosylceramides, galactosylceramides, sulfatides, and
gangliosides) due to deficiencies of enzymes required for their degradation.
b
Accumulation of mucopolysaccharides (glycosaminoglycans) due to enzyme deficiencies required for their degradation.
c
Accumulation of glycogen due to an enzyme deficiency required for its degradation.
because different enzyme deficiencies may result in the accumulation of similar or identical com-
pounds. Table 7–6 provides a list of selected lysosomal storage disorders grouped into those associ-
ated with impaired degradation of sphingolipids (the sphingolipidoses), mucopolysaccharides (the
mucopolysaccharidoses—the more recently invoked term for mucopolysaccharide is glycosamino-
glycan), and glycogen. Among the lysosomal storage diseases, Tay–Sachs, one of the sphingolipi-
doses, has been the most thoroughly studied. As a class of disorders, the lysosomal storage disorders
are rare. The one with the highest prevalence is Gaucher disease, which affects 1 in 600 in the Ashke-
nazi Jewish population. Each lysosomal storage disorder has its own characteristic clinical features,
and most signs and symptoms of these diseases are expressed early in life. Many of these disorders
can even be diagnosed in utero or in the period immediately after birth. There is often an urgency to
establish the diagnosis so that appropriate treatment can be instituted as soon as possible.
NEUROBLASTOMA
Description
Neuroblastoma is a solid tumor that affects infants and toddlers. It is the most common malig-
nancy under 1 year of age, and most patients are diagnosed by age 2 years. It presents either as a
mass, often in the abdomen or neck, or with signs and symptoms of tumor spread, including bone
pain or spinal cord dysfunction. Neuroblastoma is one of the “small round cell” tumors of child- Neuroblastoma is a
hood, and it must be distinguished from other tumors of that type, which include lymphoma, solid tumor that affects
rhabdomyosarcoma, Ewing sarcoma, and primitive neuroectodermal tumor (PNET). infants and toddlers.
It is the most common
malignancy under 1 year
Diagnosis
of age, and most patients
Measurement of urinary catecholamines can be used to alert clinicians to the likelihood that are diagnosed by age
an infant’s tumor mass is a neuroblastoma, as the other “round cell tumors” listed above do not 2 years. Measurement of
excrete catecholamines. Unlike pheochromocytoma (see Chapter 22), urinary metanephrines are urinary catecholamines
not helpful, as neuroblastomas do not produce an abundance of epinephrine. For the diagnosis can be used to alert
and monitoring of neuroblastoma, vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinicians to the likelihood
typically measured in urine by high-performance liquid chromatography (HPLC) methodology. that an infant’s tumor
The treatment of neuroblastoma patients can be followed with quantitation of VMA and mass is a neuroblastoma.
HVA in their urine. Detection of metastases in bone marrow samples of these patients is challeng-
ing with histologic methods as there are often very few tumor cells, but molecular studies are now
being used to detect minimal residual disease. Examples of such assays being used for this pur-
pose include reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase
(an enzyme involved in catecholamine synthesis) and for the proto-oncogene MYCN. The latter
gene is of interest because its copy number in neuroblastoma tumor samples has prognostic sig-
nificance. Amplification of MYCN is associated with a poorer prognosis in these patients. There is
interest in developing other biomarkers for neuroblastoma; chromogranin A and neuron-specific
enolase are 2, among several, in use or in development.
REFERENCES
Barrett PM, et al. Cystic fibrosis in an era of genomically guided therapy. Hum Mol Genet.
2012;21(R1):R66–R71.
Bastek JA, et al. The role of inflammation and infection in preterm birth. Clin Perinatol. 2011;38:385–406.
Canick J. Prenatal screening for trisomy 21: recent advances and guidelines. Clin Chem Lab Med.
2012;50:1003–1008.
DeFranco EA, et al. Improving the screening accuracy for preterm labor: is the combination of fetal fibro-
nectin and cervical length in symptomatic patients a useful predictor of preterm birth? A systematic
review. Am J Obstet Gynecol. 2013;208:233.e1–233.e6.
Lo YMD. Noninvasive fetal whole-genome sequencing from maternal plasma: feasibility studies and future
directions. Clin Chem. 2013;59:601–603.
Sandoval JA, et al. Clinical significance of serum biomarkers in pediatric solid mediastinal and abdominal
tumors. Int J Mol Sci. 2012;13:1126–1153.
Smith V, et al. A systematic review and quality assessment of systematic reviews of randomized trials
of interventions for preventing and treating preterm birth. Eur J Obstet Gynecol Reprod Biol. 2009;
142:3–11.
Tennant C, et al. Performance of lecithin-sphingomyelin ratio as a reflex test for documenting fetal lung
maturity in late preterm and term fetuses. J Matern Fetal Neonatal Med. 2012;25:1460–1462.
Valle D, et al, eds. Part 8, Amino Acids and Part 16, Lysosomal Disorders. The Online Metabolic &
Molecular Bases of Inherited Disease. New York, NY: McGraw-Hill; 2006.
C H A P T E R
Blood Vessels
Michael Laposata 8
LEARNING OBJECTIVES
1. Identify the lipid and nonlipid laboratory assays useful in the evaluation for
cardiovascular risk and describe how they are used in conjunction with other
information.
2. Learn the names of the most commonly encountered or best characterized
primary hyperlipidemias, their associated serum or plasma lipid abnormalities,
and the defects responsible for the initiation of the disorders.
3. Learn the correctable causes of hypertension that can be identified by
laboratory tests.
4. Understand the different forms of vasculitis and the role of antineutrophil
cytoplasmic antibodies (ANCA) in their diagnosis.
5. Learn the role of plasma D-dimer concentration and radiographic studies
in the diagnosis of deep vein thrombosis.
CHAPTER OUTLINE
Introduction 197 Vasculitis 204
Atherosclerosis 198 Deep Vein Thrombosis and Pulmonary
Hypertension 203 Embolism 206
INTRODUCTION
Because blood vessels are present in all organs and tissues, vascular disease is not restricted to
a limited group of signs and symptoms. All organs and tissues are potential targets of injury
in vascular disease, and most patients present with signs and symptoms indicative of injury
to a specific organ or tissue, usually as a result of diminished blood flow. For example, if there
is decreased blood flow to the heart, the patient presents with signs and symptoms related to
cardiac dysfunction. The decrease in blood flow could be the result of a lesion that originates in
the blood vessel wall, and therefore a vascular disease, or an obstruction by a blood clot inside
the blood vessel. The disorders originating within the blood vessel wall include atherosclerotic
vascular disease, hypertensive vascular disease, vasculitis, tumors, and aneurysms. Blood vessel
disorders that may result from an abnormality that is not within the blood vessel wall include deep
vein thrombosis (DVT), and what the DVT may generate if any of the clot moves to the lungs,
pulmonary embolism (PE). DVT and PE are also discussed in this chapter. A first-time DVT or
PE is nearly always the result of clot formation inside a normal vein. However, if an abnormality
in vein anatomy exists, such as congenital atresia of the inferior vena cava, such defects within the
blood vessels themselves can be highly contributory to the development of a DVT.
197
198 CHAPTER 8 Blood Vessels
• Atherosclerotic vascular disease is one of the most predominant illnesses in the Western
world. The goal of clinical laboratory testing is to identify the cause of atherosclerosis. This
is usually related to excess dietary lipid or a disorder of lipid metabolism. This chapter
provides information on disorders of lipid metabolism that lead to atherosclerosis.
• Hypertensive vascular disease is also common. The role of clinical laboratory testing is
to determine if there is a correctable cause for hypertension. Because more than 90% of
hypertension cases are “essential,” there is currently no correctable cause. Treatment with
antihypertensive medical therapy is important and beneficial, but it does not treat the
underlying cause for hypertension in most cases. Some causes of hypertension, however, are
identifiable and correctable, often surgically. An example of a surgically correctable form
of hypertension is one in which a tumor secretes a hormone responsible for the elevation
of blood pressure. Removal of the tumor typically results in normalization of the blood
pressure. The section “Hypertension” focuses on the correctable causes of hypertension
and the laboratory tests useful in identifying them.
• Vasculitis represents a less commonly encountered group of disorders with inflammation
in the blood vessel wall. Clinical laboratory testing has a limited role in establishing the
diagnosis of a particular form of vasculitis. The diagnosis is made by the specific clinical
features of the patient, the results of antineutrophil cytoplasmic antibody (ANCA) testing
for some forms of vasculitis, and, on occasion, histopathologic review of a blood vessel
biopsy specimen.
• DVT and PE are primarily diagnosed with imaging studies. However, an important test in
the clinical laboratory, used primarily to rule out DVT and PE when the result is negative,
is the D-dimer test.
ATHEROSCLEROSIS
Description
Atherosclerotic vascular disease is a major cause of mortality and morbidity in the Western world.
It is the consequence of an accumulation of lipid in large arteries including the aorta and, thereby,
a narrowing of the lumen of the arteries, which results in decreased blood flow. When an ath-
erosclerotic plaque ruptures, a thrombus can form over the ruptured plaque and totally occlude
blood flow. Atherosclerotic disease is vascular in origin in that lipid deposition and cell prolifera-
tion occur within the blood vessel wall. The end-organ damage depends on the anatomic location
of the occluded artery. It is common to have generalized atherosclerosis with multiple vascular
beds affected.
The causes of atherosclerotic vascular disease include:
• Ingestion of excess or atherogenic dietary fat, which is primarily saturated fatty acids and
cholesterol. This is the most common cause of atherosclerotic vascular disease.
• Primary lipid disorders, also known as primary hyperlipidemias, which result in an increase
in cholesterol, triglyceride, or both in the plasma. Many of these disorders are a result of
The initial approach to genetic mutations that perturb the metabolism of cholesterol. They are not uncommon.
the patient for routine • Nonlipid disorders causing elevations in the concentration of plasma lipids, usually
evaluation or for cholesterol and/or triglyceride. These are called secondary hyperlipidemias. Disorders
monitoring the status of or conditions that adversely affect lipid metabolism include hypothyroidism, nephrotic
atherosclerotic vascular syndrome, liver disease, diabetes, obesity, and alcohol abuse. In addition, many
disease is to determine medications can alter plasma lipid levels.
if the patient has an • Though not common, elevated levels of lipoprotein(a) (Lp(a) and pronounced “L-P-little a”)
elevation in serum or with or without other lipid or lipoprotein abnormalities.
plasma total cholesterol • Also not common, disorders that are associated with direct damage to the blood vessel wall,
and LDL cholesterol independent of lipid levels, such as high circulating concentrations of homocysteine.
concentrations, and a
low HDL cholesterol
concentration, and if Diagnosis
so, to first determine if The initial approach to the patient for routine evaluation or for monitoring the status of
the likely cause is excess atherosclerotic vascular disease is to determine if the patient has an elevation in serum or plasma
intake of dietary fat. cholesterol, and if so, to first determine if the cause is excess intake of dietary fat.
CHAPTER 8 Blood Vessels 199
TABLE 8–1 The Major Plasma Lipoproteins

Lipid Components Synonyms for
Lipoprotein Class of Core Lipoprotein Classes Predominant Apolipoproteinsa
Chylomicrons Triglycerides >> None B48 (also in chylomicron remnant),

cholesterol esters A-I, A-IV, C-I, C-II, C-III, E
Very-low-density Triglycerides > Pre-beta lipoproteins B-100, C-I (especially in hyperlipidemic

lipoproteins (VLDL) cholesterol esters patients), C-II (especially in
hyperlipidemic patients), C-III,
E (especially in hyperlipidemic patients)
Low-density Cholesterol esters > Beta lipoproteins B-100

lipoproteins (LDL) triglycerides
High-density Cholesterol esters > Alpha lipoproteins A-I, A-II, A-IV, C-I (especially in
lipoproteins (HDL) triglycerides normolipidemic patients), C-II (especially
in normolipidemic patients), C-III,
E (especially in normolipidemic patients)
a
Major apolipoprotein in bold.
An elevated total cholesterol level (>200 mg/dL) prompts the need to determine the plasma
or serum concentrations of low-density lipoprotein (LDL) cholesterol (a high level is bad) and
high-density lipoprotein (HDL) cholesterol (a high level is good). Table 8–1 describes the lipo-
proteins that transport lipids in the plasma.
Late in 2013, the American College of Cardiology and the American Heart Association,
in conjunction with the National Heart, Lung, and Blood Institute, developed new guidelines.
These contained substantial changes from the previous guidelines that were established more
than a decade earlier by the Adult Treatment Panel III(ATP III). Previous targets for LDL cho-
lesterol of 100 mg/dL, with the optional goal of less than 70 mg/dL, were removed as indicators
of treatment success. Instead, 4 treatment groups for statin therapy have been identified. They
are individuals with clinical atherosclerotic vascular disease; those with LDL cholesterol levels
greater than 190 mg/dL; those with diabetes between 40 and 75 years old with LDL cholesterol
levels between 70 and 189 mg/dL without evidence of atherosclerotic vascular disease; and indi-
viduals without evidence of cardiovascular disease or diabetes who have LDL cholesterol levels
between 70 and 180 mg/dL and a 10-year risk of atherosclerotic vascular disease greater than
7.5%. The risk for stroke has been added to the coronary events traditionally covered by car-
diovascular risk assessments. Specific recommendations on lifestyle for cardiovascular disease
prevention include eating a diet rich in fruits, vegetables, whole grains, fish, low-fat dairy, lean
poultry, nuts, legumes, non-tropical vegetable oils with restriction of saturated fats, trans fats,
sweets, sugar sweetened beverages, and sodium; and engaging in aerobic physical activity of
moderate to vigorous intensity lasting 40 minutes per session 3 to 4 times per week. With regard
to weight loss, the new recommendation is that patients with a BMI of 25, not 30 as in the past,
who have even one comorbidity, such as an elevated waist circumference, should begin treatment
for weight loss.
LDL Cholesterol
The most common method for determining LDL cholesterol is a calculation that requires the use
of a fasting sample with a triglyceride level less than 400 mg/dL. The LDL is calculated accord-
ing to the Friedewald formula, which is: calculated LDL cholesterol = total cholesterol − HDL
cholesterol − (triglycerides/5). The very-low-density lipoprotein (VLDL) fraction is represented
by (triglycerides/5) with the assumption that there is very little triglyceride in LDL and HDL.
Because the plasma and serum triglyceride concentration increases with ingestion of dietary fat,
a fasting sample is required for an accurate calculation of LDL cholesterol using the Friedewald
formula. Although it is acceptable to drink water, the patient may not ingest any calories 8 to
12 hours before the blood sample is collected. If the patient does not fast, and the triglyceride
is elevated above baseline, the calculated LDL cholesterol will be falsely low. Another challenge
to an accurate determination of LDL cholesterol is that there is substantial biological variability,
independent of any change in dietary habits, in the total cholesterol level. This would also have a
significant impact on the calculated LDL value. For that reason, testing for total cholesterol and
LDL cholesterol should be repeated on a second sample drawn 1 to 8 weeks later. The mean value
from these 2 samples is used, as long as the differences between them are less than 30 mg/dL in
total cholesterol. If the difference is greater than 30, a third sample should be obtained and the
mean of the 3 samples calculated. The day-to-day variability in total cholesterol within a single
individual is typically at least 10% and can be as high as 30%. This level of variability, in a patient
whose LDL cholesterol is calculated using the Friedewald formula, could span a range of values
from 125 to 165 mg/dL if the patient has a true value of 145 mg/dL. The Friedewald calculation
fails if the triglyceride concentration in the fasting sample is higher than 400 mg/dL. At such high
concentrations, the (triglyceride concentration/5) is no longer a reasonable estimate of the VLDL
cholesterol concentration.
Assays that directly measure LDL are also available. These are not dependent on the Friedewald
formula, and therefore independent of the triglyceride concentration. For that reason, fasting is
not required if a direct LDL assay is performed. The direct LDL cholesterol assay circumvents the
issues associated with the calculated LDL cholesterol when it is greater than 400 mg/dL. However,
even though these assays are routinely used, some studies have shown substantial imprecision
with the assay, although this conclusion has been challenged by other studies. In addition, direct
LDL cholesterol measurement is not useful in patients with a dyslipidemia.
HDL Cholesterol
Low levels of HDL cholesterol (<40 mg/dL) represent a cardiac risk factor. However, an elevated
HDL cholesterol concentration greater than or equal to 60 mg/dL reduces cardiovascular risk, and
if present, it allows subtraction of 1 risk factor from the sum of the risk factors. The patient does
not need to fast prior to sample collection for performance of the HDL cholesterol.
Total Cholesterol
The total cholesterol concentration is the sum of HDL cholesterol, LDL cholesterol, VLDL
cholesterol, intermediate-density lipoprotein cholesterol (IDL cholesterol), and cholesterol
associated with LP(a). In the vast majority of patients, the cholesterol in IDL and in LP(a) is very
small relative to the other lipoproteins. The patient does not need to fast prior to sample collection
for performance of the total cholesterol. Like HDL cholesterol, total cholesterol is not affected by
recent dietary intake.
Non-high-density Lipoprotein Cholesterol

Non-HDL cholesterol is the difference between the total cholesterol concentration and the
HDL cholesterol concentration. The remaining lipoprotein particles—LDL, VLDL, IDL, and
LP(a)—are all atherogenic. In some clinical trials, non-HDL cholesterol was shown to be
superior over LDL cholesterol as a measurement of cardiovascular risk. Because neither the
total cholesterol nor the HDL cholesterol is affected by the triglyceride level or ingestion of
dietary fat, non-HDL cholesterol can be calculated on nonfasting specimens. Use of non-HDL
cholesterol measurements instead of calculated LDL cholesterol measurements avoids the
problem of calculating LDL cholesterol in patients who have triglyceride concentrations greater
than 400 mg/dL.
High-sensitivity C-reactive Protein

The level of persistent inflammatory processes relates to the risk of cardiovascular events. Patients
who have an existing inflammatory process typically have CRP levels greater than 3.0 mg/L.
Transient elevations in CRP associated with benign processes occur frequently, and for that reason
repeat testing within 2 weeks of an elevated value is recommended to determine if the CRP level
exceeds 3.0 mg/L. The American Heart Association and the National Cholesterol Education Panel
concur that CRP levels should be measured only after traditional lipid parameters have been
assessed completely, with the CRP levels used to classify only those patients who are considered
to have borderline cardiovascular risk. Values for CRP of less than 1.0 mg/L represent low risk;
1.0 to 3.0 mg/L is intermediate cardiovascular risk; in patients with greater than 3.0 mg/L CRP
is considered to be at high cardiovascular risk. The CRP assay was not originally designed for a
high-sensitivity analysis, and previously was only used to measure much higher values than the
levels used in cardiovascular risk assessment.
Tests Not Included for Cardiovascular Risk by the 2010 American College
of Cardiology Foundation/American Heart Association Task Force
on Practice Guidelines for Asymptomatic Individuals
Homocysteine, LP(a) (primarily because of lack of standardized assays), fibrinogen (although
immunoassays appear to provide better predictive data than functional clot-based assays), apo-
lipoproteins (even though low apo A-I levels, associated with HDL, and high apo B-100 levels,
associated with LDL, are found in patients with increased cardiac risk, neither parameter adds
information beyond what is provided by HDL cholesterol and LDL cholesterol), lipoprotein
particle size, lipoprotein density, natriuretic peptides, and genomic testing.
Metabolic Syndrome
Metabolic syndrome is present when a series of risk factors for developing heart attack stroke
and diabetes are present. In the United States, metabolic syndrome is extremely common,
affecting more than 40% of people above the age of 60. Patients with the metabolic syndrome
do not have physical symptoms. However, patients who have the metabolic syndrome, over
time, commonly develop atherosclerotic vascular disease (myocardial infarction and stroke),
kidney dysfunction, and type 2 diabetes with its associated risks and complications. Different
expert groups have established the risk factors and their levels that are part of the metabolic
syndrome from the American Heart Association and the National Cholesterol Educational
Program.
Waistline: For men, greater than 40 in; for women, greater than 35 in
Elevated triglycerides: Equal to or greater than 150 mg/dL
Reduced HDL cholesterol: For men, less than 40 mg/dL; for women, less than 50 mg/dL
Elevated blood pressure: Equal to or greater than 130/85 mm Hg or use of a medication
for hypertension
Elevated fasting glucose: Equal to or greater than 100 mg/dL or use of a medication
for hyperglycemia
Assessment of Cardiovascular Risk

Patients for whom testing for lipid status and inflammation is most useful include those with
factors that increase cardiovascular risk, such as cigarette smoking, hypertension, diabetes
mellitus, obesity, physical inactivity, or a family history of coronary heart disease, who are
currently asymptomatic and have no history of coronary heart disease. As noted below in
the scoring calculations for cardiovascular risk, decisions about patient management involve
an assessment for both non-laboratory-test- and laboratory test-associated risk factors. There
are a number of risk calculations based on a combination of data from clinical history, signs
and symptoms, and laboratory test results. The following are risk scores and their associated
parameters. Collectively, they represent a risk for developing atherosclerotic plaques and to a
much lesser extent, with inflammatory markers included, the risk of plaque rupture.
The Framingham score: total cholesterol, HDL cholesterol, age, gender, smoking status,
and blood pressure.
The Reynolds score: high-sensitivity C-reactive protein (hs-CRP), total cholesterol, HDL
cholesterol, age, gender, parental history, smoking status, and blood pressure. In women
with diabetes, the hemoglobin A1C result is also included.
The PROCAM score: LDL cholesterol, HDL cholesterol, triglycerides, family history of
coronary artery disease, smoking status, age, and presence of diabetes. This score is
validated only in men.
Systematic coronary risk evaluation (SCORE): total cholesterol, HDL cholesterol, age,
gender, smoking status, and blood pressure.
Genetic Disorders Classified by Causation of an Excess

or Deficiency of a Specific Class of Lipoprotein
Genetic abnormalities are much less common explanations for high blood lipid levels than excess
intake of dietary fat.
• LDL-associated abnormalities:
Familial hypercholesterolemia: Defects in the LDL receptor gene cause an accumulation
of LDL particles in the plasma. Patients have an elevated plasma LDL cholesterol and
premature coronary artery disease.
Familial defective apolipoprotein B: The defective apolipoprotein B has a reduced affinity
for the LDL receptor. Affected individuals usually have an elevated LDL cholesterol,
but there is wide variability in the LDL cholesterol levels.
Hypobetalipoproteinemia: Hypobetalipoproteinemia results from a mutation within
the apolipoprotein B gene that results in truncation of the mature apolipoprotein B.
This condition is not associated with an increased risk of cardiovascular disease.
Abetalipoproteinemia: This condition is caused by mutation in a gene coding for a protein
required for assembly of apolipoprotein B-containing lipoproteins in the plasma.
Affected patients suffer from mental and developmental retardation as children.
• Triglyceride-rich lipoprotein (VLDL and chylomicrons [CM]-associated) abnormalities:
Familial hypertriglyceridemia: This disorder is highly heterogeneous and likely results
from several genetic defects with a strong environmental influence. Plasma triglycerides
and VLDL cholesterol are moderately to markedly elevated. There is not a strong
relationship with coronary artery disease.
Familial hyperchylomicronemia: These patients have severe hypertriglyceridemia with
associated complications such as pancreatitis, xerostomia, and xerophthalmia. The
hypertriglyceridemia results from either a markedly reduced or absent level of lipoprotein
lipase activity or, less commonly, the absence of the activator of this enzyme, which is
apolipoprotein C-II.
Type III hyperlipoproteinemia: This disorder is characterized by an accumulation in
the plasma of remnant lipoprotein particles, mostly from CM and VLDL. The excess
plasma lipoprotein results in pathognomonic tuberous xanthomas and palmar striated
xanthomas.
Familial combined hyperlipidemia: This is a common familial lipoprotein disorder strongly
associated with coronary artery disease. The disorder appears to result from hepatic
overproduction of apolipoprotein B-containing glycoproteins, delayed clearance of
triglyceride-rich lipoprotein, and increased flux of free fatty acids to the liver. There is
genetic heterogeneity among patients with this disorder and considerable overlap exists
between this disorder and metabolic syndrome.
• HDL-associated abnormalities:
Apolipoprotein A-1 gene defects: Defects in genes for apolipoprotein A-I, C-III, and A-IV
can all decrease the production of HDL particles. Some of these defects are associated
with premature cardiovascular disease. However, other mutations appear to confer
longevity despite very low HDL levels.
Lecithin cholesterol acyltransferase (LCAT) deficiency: A deficiency of LCAT results in
reduced conversion of cholesterol to cholesterol esters in the plasma. This results in low
HDL cholesterol levels, corneal opacities, and hemolytic anemia.
Cholesterol ester transfer protein (CETP) deficiency: Patients with this deficiency have
extremely elevated HDL cholesterol levels, which are primarily cholesterol esters. A
deficiency of this enzyme causes accumulation of cholesterol esters within the HDL
particles, and, therefore, this deficiency is not associated with premature coronary artery
disease.
Tangier disease: Individuals with this disorder have reduced cellular cholesterol efflux.
The mutation is in the gene that codes for the protein known as ATP-binding cassette
A1 (ABCA1), which is a transporter protein. Many different mutations within this gene
have been associated with Tangier disease, which is associated with an increased risk
for coronary artery disease and extremely low HDL levels.
Familial HDL deficiency: These patients have low HDL cholesterol levels and are at
increased risk for coronary artery disease. This is more common than Tangier disease
and also appears to result from mutations in the ABCA1 gene.
HYPERTENSION A commonly accepted

definition of hypertension
Description is a systolic reading
Hypertension is a very common chronic disease, particularly in Western countries. In the United >140 mm Hg or a diastolic
States, approximately 1 in 3 adults suffers from hypertension, but most of these individuals have reading >90 mm Hg on
no identifiable cause for their hypertension. A number of clinical practice guidelines have been at least 3 occasions, with
a minimum of 2 weeks
published that provide a definition of hypertension. Although there is some variability, a normal
between each of the
systolic blood pressure is between 120 and 129 mm Hg, and a normal diastolic blood pressure
3 readings.
is between 80 and 84 mm Hg. Systolic blood pressures between 130 and 139 and diastolic blood
pressures between 85 and 89 are considered high normal by most guidelines; mild hypertension is
systolic blood pressures of 140 to 159 and diastolic blood pressures of 90 to 99; systolic blood pres-
sures of 160 to 179 or diastolic blood pressures of 100 to 109 represent moderate hypertension; and
severe hypertension is defined as systolic blood pressure greater than 180 or diastolic blood pres-
sure greater than 110. (The 2013 reference by Al-Ansary et al is a systematic review of the recent
clinical practice guidelines on the diagnosis, assessment, and management of hypertension.)
In evaluating a patient with hypertension, 1 question is whether there is an identifiable cause
for the hypertension or whether it is idiopathic or “essential.” Another important question is
whether the hypertension has resulted in damage to the organs commonly injured in hyperten-
sive individuals, namely, the brain, the heart, and the kidneys.
Hypertension with a potentially correctable cause is suggested by certain symptoms, such as
flushing and sweating (which are associated with pheochromocytoma), findings on physical exam-
ination such as a renal bruit (which is associated with renal artery stenosis), and laboratory abnor-
malities such as hypokalemia, which can be found in patients with hyperaldosteronism. Among
children, up to 85% with hypertension have a secondary cause and, therefore, require a careful eval-
uation of the patient for correctable cause. In children up to the age of 18 years, coarctation of the
aorta and renal parenchymal disease are common etiologies for hypertension. Among adults, 5%
to 10% for patients with hypertension have a secondary cause. Young adults with secondary hyper-
tension are often explained by abnormalities in thyroid function and fibromuscular dysplasia. In
middle-age adults, the most common secondary cause of hypertension is hyperaldosteronism. In
older adults, atherosclerotic renal artery stenosis, renal failure, and hypothyroidism predominate
as secondary causes. There are many drugs causative for an elevation in blood pressure in some
patients. Notable ones include oral contraceptives, selected nonsteroidal anti-inflammatory drugs
such as ibuprofen, a variety of drugs used to treat psychiatric disorders such as tricyclic antidepres-
sants, steroids such as methylprednisolone, and many herbal medicines and illicit drugs.
To understand the causes of hypertension, it is necessary to understand the mechanism
by which blood pressure is regulated (see Chapter 22 for a related discussion on adrenal gland
hormones). In response to decreased arterial pressure from a variety of causes, there is decreased
blood flow to the kidney, causing the kidney to secrete renin. Renin released within the renal
circulation converts angiotensinogen to angiotensin I, which is subsequently converted to
angiotensin II. This molecule acts on the adrenal cortex to release aldosterone. Aldosterone
increases sodium retention by the kidney, and thereby expands the extracellular fluid volume
and returns the blood pressure to normal. Any alteration in this pathway, such as an increase in
aldosterone or a decrease in blood flow to the kidney, will activate the renin–angiotensin system,
lead to inappropriate fluid accumulation, and increase the blood pressure. This is why many of
the diseases producing hypertension listed in Table 8–2 are associated with kidney dysfunction.
The renal disorders that are associated with hypertension can be renovascular, in which case
the blood flow to the kidney is decreased, or they can be parenchymal. Parenchymal diseases
include chronic kidney infections, glomerulonephritis, and polycystic kidney disease, among
many others. Most of these conditions are treatable, and treatment of the underlying disorder
may reduce the hypertension. Abnormalities in the adrenal gland, such as a pheochromocytoma
TABLE 8–2 Evaluation of the Patient for Hypertension to Determine if Hypertension Is Essential or Correctable
Disorder Tests Results Supporting the Diagnosis Results
Drug-induced hypertension Positive history for ingestion of sympathomimetics, corticosteroids, mineralocorticoids,

vasopressin, or cocaine, among other drugs, which have a hypertensive effect
Renal and vascular causes of hypertension
Renal artery stenosis Angiography and imaging studies consistent with stenosis; increase in serum creatinine after
starting angiotensin-converting enzyme inhibitor or angiotensin receptor blocker; renal bruit
Chronic renal disease of multiple etiologies Elevated BUN and creatinine
Polycystic kidney disease Radiologic studies that confirm cystic disease of the kidney
Renin-secreting tumors (renal or extrarenal) Elevated plasma renin activity, normal renal angiogram, low serum potassium, and elevated
urinary aldosterone secretion
Coarctation of the aorta (decreased blood flow Imaging studies; arm to leg blood pressure difference >20 mm Hg
to kidney resulting from a defect in the aorta)
Adrenal causes of hypertension

Primary hyperaldosteronism Low or borderline serum potassium and elevated urinary aldosterone secretion
17-Alpha hydroxylase deficiency Reduction in activity of 17-alpha hydroxylase (see Chapter 22); similar to primary
hyperaldosteronism but with virilization and precocious puberty in males
11-Beta hydroxylase deficiency Reduction in activity of 11-beta hydroxylase (see Chapter 22); similar to primary
hyperaldosteronism but with virilization and precocious puberty in males
Cushing syndrome Test results consistent with one of the different forms of Cushing syndrome
(see Chapter 22 for diagnostic tests)
Pheochromocytoma Test results that demonstrate an excess of catecholamines (see Chapter 22 for diagnostic tests)
Thyroid disorders Abnormal thyroid-stimulating hormone (TSH) test as a screening assay for thyroid function
BUN, blood urea nitrogen.
or an aldosterone-secreting tumor (discussed in Chapter 22), also can lead to hypertension, and
may be surgically correctable.
Diagnosis
The hypertensive patient undergoing evaluation is first studied using a number of routine tests
including:
• Complete blood count to determine if the patient is anemic or polycythemic.
• Electrolyte measurements to measure the potassium and bicarbonate levels.
• Creatinine concentration in plasma or serum and creatinine clearance to assess renal
function.
• Glucose (usually a fasting level) to diagnose diabetes, because diabetic patients have an
approximately 2-fold higher incidence of hypertension than nondiabetic patients.
• Urinalysis to detect the presence of diabetes by glucose in the urine; urinalysis also may
indicate the presence of significant parenchymal disease in the kidney if proteinuria,
hematuria, or pyuria is present.
Inflammation in the blood
vessel wall is known The tests to further investigate the cause of hypertension beyond the screening tests are more
as vasculitis. The large invasive, costly, or esoteric. These are noted in Table 8–2.
number of different
vasculitides, which are
sometimes overlapping in VASCULITIS
their clinical or anatomic
characteristics, often Description
makes the diagnosis of a The systemic vasculitides are disorders in which there is inflammation of the blood vessels and
specific form of vasculitis tissue necrosis. The different forms of vasculitis are classified by the size of the vessels affected.
challenging. The classification of the different forms of vasculitis remains controversial and suboptimal, largely
because of the low prevalence of these diseases, which are anatomically, epidemiologically, and
clinically distinct. Vasculitis is known as primary vasculitis when there is no identifiable underly-
ing etiology. Secondary vasculitis represents vasculitides in which there is an underlying condi-
tion. The underlying condition may be an infection such as HIV, or hepatitis B or hepatitis C,
or an underlying connective tissue disease such as lupus or rheumatoid arthritis. The basis for
identifying a specific form of vasculitis is histopathologic examination of the blood vessel, but this
is often not feasible because of the blood vessels involved and the danger of obtaining tissue from
them. It is for this reason that the classification must rely on criteria other than histopathology.
The large number of different vasculitides, which are sometimes overlapping in their clinical
or anatomic characteristics, often makes the diagnosis of a specific form of vasculitis challenging.
In general, a diagnosis is made by 1) the presence of characteristic clinical findings for the par-
ticular form of vasculitis and 2) inflammation within a specific size of blood vessels, as shown in
Table 8–3. There are vasculitides that are infectious in origin that are not included in Table 8–3.
Rocky Mountain spotted fever, syphilis, aspergillosis, herpes, and neisserial infections can all be
associated with vasculitis. (See Chapter 5 for information on organisms and infections that can
cause vasculitis.)
Diagnosis
The laboratory testing is different for each of the vasculitides listed in Table 8–3. ANCA are
autoantibodies, typically IgG, directed against antigens in neutrophils (most commonly) and
monocytes. Because they are detected in patients with some forms of systemic vasculitis, known
as the ANCA-associated vasculitides, they have been used diagnostically to identify patients with
these particular forms of systemic vasculitis. These include Wegener’s granulomatosis, microscopic
TABLE 8–3 Laboratory Evaluation for Selected Noninfectious Causes of Vasculitis

Vasculitic Disorder Vessels With Inflammation Clinical Laboratory Testing
Giant cell (temporal) arteritis Aorta and large- to medium-sized Elevated erythrocyte sedimentation rate (ESR) or C-reactive protein
arteries (CRP) in most patients
Takayasu arteritis Aorta and large- to medium-sized Elevated ESR or CRP in most patients; BUN, creatinine, and urinalysis
arteries to assess and monitor renal disease
Polyarteritis nodosa Medium-sized arteries; small arteries Small aneurysms strung like beads of a rosary making the “rosary sign”;
without pulmonary or glomerular no specific lab tests
involvement
Kawasaki disease Large- to medium-sized arteries; Laboratory testing is not informative with self-limited form of the
small arteries disease; if cardiac complications occur, tests for damage to cardiac
muscle may be useful (see Chapter 9)
Wegener’s granulomatosis Small arteries, arterioles, capillaries, Antiproteinase 3 (anti-PR3) ANCA (c-ANCA) detectable in the large
venules, veins majority of patients with active disease; a much smaller percentage
have antimyeloperoxidase (anti-MPO) ANCA (p-ANCA)
Churg–Strauss syndrome Small arteries, arterioles, capillaries, Antimyeloperoxidase ANCA detectable in most patients; eosinophilia
venules, veins
Microscopic polyangiitis Small arteries, arterioles, capillaries, Antimyeloperoxidase ANCA (more common) or antiproteinase
venules 3 ANCA (less common) detectable in most cases; BUN, creatinine,
and urinalysis to assess and monitor renal abnormalities
Henoch–Schönlein purpura Arterioles, capillaries, venules; BUN, creatinine, and urinalysis to assess and monitor renal
immune complex-mediated abnormalities; palpable purpura from small hemorrhages
vasculitis, involving IgA
Essential cryoglobulinemic Arterioles, capillaries, venules; Serum cryoglobulin with identification of type and quantitation,
vasculitis immune complex-mediated if present (see discussion on cryoglobulinemia in Chapter 3)
vasculitis caused by cryoglobulins
Cutaneous leukocytoclastic Capillaries, venules, arterioles May have underlying autoimmune, neoplastic, or infectious process
angiitis or an accompanying vasculitis of a different type; laboratory testing
is directed at detection of underlying diseases
ANCA, antineutrophil cytoplasmic antibody; BUN, blood urea nitrogen.
polyangiitis, and Churg–Strauss syndrome as shown in Table 8–3. Immunofluorescence on

ethanol-fixed neutrophils helps to differentiate the different ANCA patterns. Although there are
subtypes, the principal forms of staining are p-ANCA (which is perinuclear staining), c-ANCA
(which is cytoplasmic staining), and atypical ANCA. The most common target of p-ANCA
antibodies is myeloperoxidase, a neutrophil granule protein involved in the generation of oxygen
radicals. Less commonly these antibodies will recognize lactoferrin, elastase, and cathepsin G.
The c-ANCA antigen is specifically proteinase 3 (PR3).
Some of the vasculitides will affect the kidney, and for those, monitoring of renal function is
important. The diagnosis of a particular form of vasculitis can be supported by a variety of other
test results.
DEEP VEIN THROMBOSIS AND PULMONARY EMBOLISM

Description
DVT and PE are common disorders. The major concern for patients with DVT is the risk of embo-
lism to the lungs (PE). The presence of a thrombosis in a deep vein in the leg is a risk factor for PE,
but a thrombosis in a superficial vein in the leg is not. Clots in the superficial veins cannot embolize
to the lungs. A DVT in the leg that is above the knee presents a significantly greater risk for PE than
does a thrombosis in a deep vein that is below the knee. If the DVT has extended above the knee,
patients are more likely to experience soft tissue swelling and discomfort, distention of the vein (a
palpable “cord” on physical examination), Homans sign (pain on dorsiflexion of foot), erythema,
and warmth. Upper extremity (usually arm) DVTs are much less common than lower extremity
(leg) DVTs. A lower extremity DVT, especially if it is small, is often asymptomatic. Thrombosis
in the pulmonary circulation can occur independently of DVT, but thrombosis in the pulmonary
vasculature commonly results from thrombi originally developed in the deep veins of the leg.
The major concern for Both DVT and PE are commonly associated with one or more congenital or acquired risk
patients with DVT is factors for thrombosis. The acquired factors include trauma, immobilization, the postoperative
the risk of embolism state, antiphospholipid antibodies, malignancy, myeloproliferative disorder, pregnancy, and the
to the lungs. A lower postpartum state, among many others. The most commonly encountered congenital risk factors,
extremity DVT, especially described in detail in the section “Hypercoagulable States” in Chapter 11, include the factor V
if it is small, is often Leiden mutation that produces activated protein C resistance, the prothrombin G20210A muta-
asymptomatic. tion, and deficiencies of protein C, protein S, and antithrombin.
Diagnosis
As a thrombus is degraded, degradation products of cross-linked fibrin are generated. One of
these degradation products is the D-dimer. The D-dimer levels are typically elevated in patients
with DVT and PE. However, the D-dimer level can be elevated in many other clinical conditions
associated with fibrin formation and degradation. These include malignancy, trauma, disseminated
intravascular coagulation, and the postoperative states. Because of this, the diagnostic strength
of the D-dimer test is its effectiveness in ruling out DVT and PE, when the result is negative.
The assays for D-dimer measurement involve the use of monoclonal antibodies that specifically
recognize the D-dimer. There are many different clinical assays with different sensitivities and
specificities, but despite the variability in available assays for D-dimer, there are now many that
can be used effectively in ruling out thrombosis in patients who do not have a high clinical
probability of DVT and PE.
Clinical decision rules have been useful in standardizing the evaluation of patients
with suspected DVT or PE, before the determination of the D-dimer. One commonly used
clinical decision rule is the Wells rule for DVT. Information from the medical history and
physical examination are obtained, and points are assigned based on the presence of these
individual parameters. For PE, 2 extensively validated clinical decision rules are the Wells rule
and the revised Geneva score. These 2 decision rules vary in the items that are included in
the evaluation and the number of points assigned when the individual parameters are present.
Imaging studies provide definitive diagnostic information in the evaluation of patients for
DVT and for PE. In patients suspected of acute DVT, with a clinical decision rule score that
makes DVT unlikely, the D-dimer test is performed first. If the test is negative, DVT is ruled out,
TABLE 8–4 Tests for the Diagnosis of Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE)
Tests for DVT or PE Description Advantages Disadvantages Comments
Compression Vein is visualized by ultrasound; then Useful for symptomatic Less sensitive for Most commonly used
ultrasonography external compression is applied to the proximal lower extremity DVT; asymptomatic DVT imaging procedure for
(for DVT) skin surface above the vein; normal noninvasive; highly specific and distal lower initial evaluation of the
veins collapse and thrombosed veins extremity DVT patient suspected of
are not compressible having a DVT
Computed X-rays are used to generate Convenient for diagnosing Intravenous contrast
tomography (CT) 3-dimensional images of the body pulmonary embolism material required
scan for PE
Computed Filling defect is shown on delayed Correlates well with Radiation dose, cost, For patient with leg
tomography (CT) contrast-enhanced scan ultrasonography and good and scanning time swelling and negative
scan for DVT diagnostic accuracy or uncertain ultrasound
results
D-dimer D-dimer is a specific degradation Simple blood test in a patient A positive result has Insensitive methods
measurement product of cross-linked fibrin that is with a low risk for DVT or PE; to be confirmed including manual latex
(for ruling out produced by physiologic fibrinolysis using a sensitive method, a by a more specific agglutination must not
DVT and PE) of thrombi negative D-dimer test has a imaging test be used to rule out DVT
high negative predictive value or PE
in excluding DVT and PE
but if the test is positive, a compression ultrasonography study is performed. A positive com-
pression ultrasonography test confirms the DVT, and a negative test rules out DVT. In patients
who have an evaluation with clinical decision rules that makes a DVT likely, the compression
ultrasound is performed before the D-dimer. A positive compression ultrasound confirms the
DVT, but a negative compression ultrasound prompts the performance of the D-dimer. A nega-
tive D-dimer test rules out the DVT, but a positive D-dimer test prompts a repeat compression
ultrasound in 1 week. Computed tomography (CT) scanning for DVT can be used to evaluate the
patient with leg swelling and equivocal compression ultrasonography.
Patients suspected of acute PE but with an unlikely diagnosis by clinical decision rules are
evaluated first with a D-dimer test, just like for DVT. A negative D-dimer rules out the PE, but
a positive result leads to the performance of a CT pulmonary scan. This imaging study involves
injection of intravenous contrast material. The presence of intraluminal filling defects in the pul-
monary arteries confirms a diagnosis of PE. In patients suspected of acute PE whose clinical deci-
sion rule analysis suggests PE, the imaging study is performed without the D-dimer test, and the
result of the imaging study determines whether the PE is present or absent.
There are a variety of other imaging studies that can be performed to evaluate patients for
DVT or PE, but these are much less commonly used. PE is also discussed in Chapter 14.
Table 8–4 presents the imaging studies and the D-dimer tests and their use in the diagnosis
of DVT and PE.
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C H A P T E R
The Heart
Fred S. Apple 9
LEARNING OBJECTIVES
1. Learn the differential diagnosis of ischemic chest pain and the laboratory
tests used in the assessment of myocardial injury, including acute
myocardial infarction.
2. Learn the clinical features of congestive heart failure (CHF) and the laboratory
tests useful in ruling in and ruling out CHF and monitoring and risk outcomes
assessment of patients with this disorder.
CHAPTER OUTLINE
Introduction 209 Orders for Serial Cardiac Troponin
Acute Myocardial Infarction 209 Testing 215
Description 209 High-sensitivity Cardiac Troponin
Diagnosis 210 Assays 215
Cardiac Troponin 212 Congestive Heart Failure 216
Analytical Methods for Measuring Description 216
Cardiac Troponin 213 Diagnosis 216
99th Percentile Reference Value as Implications for Therapy Using Test Results
a Cutoff for Diagnosis of Acute for Natriuretic Peptides 218
Myocardial Infarction 214 Biological Variability 218
Role of Cardiac Troponin for Risk Novel Biomarkers for Heart Failure Risk
Outcomes Assessment 214 Assessment 218
INTRODUCTION
There are many forms of cardiac disease. This chapter briefly covers the role of biomarkers in
acute myocardial infarction (AMI) and congestive heart failure (CHF). The large numbers of
other cardiac diseases are not discussed in this chapter because of the relatively minor role of
diagnostic clinical laboratory tests in these disorders.
ACUTE MYOCARDIAL INFARCTION

Description
The term AMI is defined as an imbalance between myocardial oxygen supply (ischemia) and
demand, resulting in injury to and the eventual death of myocytes. AMI should be used when
there is evidence of myocardial necrosis in a clinical setting consistent with acute myocardial
ischemia. Such necrosis is most often associated with a thrombotic occlusion superimposed on
coronary atherosclerosis. It is now apparent that the process of plaque rupture and thrombosis
is 1 of the ways in which coronary atherosclerosis progresses. Total loss of coronary blood flow
209
210 CHAPTER 9 The Heart
results in a clinical syndrome associated with an ST-segment elevation MI (STEMI). Partial loss
of coronary perfusion, if severe, can lead to necrosis as well, which is generally less severe and is
known as non-ST-segment elevation MI (NSTEMI). Both STEMI and NSTEMI are considered
type 1 MIs. In instances of myocardial injury with necrosis with a condition other than coro-
nary artery disease (CAD), which contributes to an imbalance between oxygen supply and/or
demand (eg, coronary endothelial dysfunction, respiratory failure, hypotension, etc), this MI
is a type 2 MI that is secondary to ischemic imbalance. Other ischemic events of lesser severity
without myocardial necrosis are designated as angina, which can range from stable to unstable.
About 1.7 million patients are hospitalized each year in the United States with an acute coro-
nary syndrome (ACS). Approximately 700,000 patients suffer from an initial AMI annually and
another 500,000 from a recurrent AMI. Coronary heart disease causes 20% of all deaths and
cardiovascular diseases up to 40%. Historically, most deaths caused by ischemic heart disease
have been acute, but as our therapeutic abilities have improved, the disease is slowly becoming
a more chronic one.
In many patients with AMI, no precipitating factor can be identified. The clinical history
remains of substantial value in establishing a diagnosis. A prodromal history of angina can be
elicited in 40% to 50% of patients with AMI. Of the patients with AMI presenting with prodromal
symptoms, approximately one third have had symptoms from 1 to 4 weeks before hospitalization;
in the remaining two thirds, symptoms predate admission by a week or less, with one third hav-
ing had symptoms for 24 hours or less. The pain of AMI is variable in intensity, and the discom-
fort is described as a squeezing, choking, vise-like, or heavy pain. It may also be characterized
as a stabbing, knife-like, boring, or burning discomfort. Often the pain radiates down the left
arm. In some instances, the pain of AMI may begin in the epigastrium and simulate a variety
of abdominal disorders, which often causes AMI to be misdiagnosed as indigestion. In other
patients, the discomfort of AMI radiates to the shoulders, upper extremities, neck, and jaw, again
usually favoring the left side.
Diagnosis
The ideal biomarker of myocardial injury should 1) provide early detection of myocardial
injury, 2) provide rapid, sensitive, and specific diagnosis for an AMI, 3) serve as a risk
stratification tool in ACS patients, 4) assess the success of reperfusion after thrombolytic
therapy, 5) detect reocclusion and reinfarction, 6) determine the timing of an infarction and
infarct size, and 7) detect procedural-related perioperative MI during cardiac or noncardiac
surgery. In reality, no 1 biomarker is able to 100% cover all these areas. However, cardiac
troponin (cTn) does provide the power to be utilized in the majority of these clinical areas.
Ruling in AMI requires a test with high diagnostic sensitivity (preferred by the ER physician
in the urgent care, emergency setting as to not send anyone home with an AMI), whereas
ruling out AMI requires a test with high diagnostic specificity (preferred by the cardiologist
following admission to avoid excessive and costly diagnostic evaluations in the non-AMI
patient). It is the function of the laboratory to provide advice to physicians about cardiac
biomarker/troponin characteristics.
An updated 2012 “Global Until 2000, the diagnosis of AMI established by the World Health Organization (WHO)
Task Force for the Third required at least 2 of the following criteria: 1) a history of chest pain, 2) evolutionary changes
Universal Definition of MI”
on the ECG, and/or 3) elevations of serial cardiac biomarkers (total creatine kinase [CK] and
has codified the role of
CKMB). It was rare for a diagnosis of AMI to be made in the absence of biochemical evidence of
biomarkers. The advocate
myocardial injury. A 2000 ESC/ACC consensus conference, updated in 2007 and most recently
that the diagnosis be
made from evidence of in 2012 by the “Global Task Force for the Third Universal Definition of MI,” has codified the
myocardial injury based role of biomarkers. The advocate that the diagnosis be made from evidence of myocardial injury
on biomarkers of cardiac based on biomarkers of cardiac damage, preferably cTnI or cTnT, in the appropriate clinical
damage, preferably situation of ischemic symptoms (Table 9–1). This guideline does not suggest that all increases
cardiac troponin (cTn) of cTn should elicit a diagnosis of AMI, but only those associated with the appropriate clinical,
I or T, in the appropriate ECG, and imaging findings. When cTn increases that are not caused by acute ischemia occur,
clinical situation of the clinician is obligated to search for another etiology for the elevation, a number of which
ischemic symptoms. are shown in Table 9–2. The initial ECG is diagnostic of AMI in about 30% of AMI patients.
CHAPTER 9 The Heart 211
TABLE 9–1 Criteria for Diagnosis of Acute Myocardial Infarction

The term acute myocardial infarction (MI) should be used when there is evidence of myocardial necrosis in a clinical setting consistent with acute
myocardial ischemia. Under these conditions any 1 of the following criteria meets the diagnosis for MI:
• Detection of a rise and/or fall of cardiac biomarker values (preferably cardiac troponin [cTn]) with at least 1 value above the 99th percentile upper
reference limit (URL) and with at least 1 of the following:
° Symptoms of ischemia
° New or presumed new significant ST-segment T-wave (ST-T) changes or new left bundle branch block (LBBB)
° Development of pathological Q waves in the ECG
° Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality
° Identification of an intracoronary thrombus by angiography or autopsy
• Cardiac death with symptoms suggestive of myocardial ischemia and presumed new ischemic ECG changes or new LBBB, but death occurred
before cardiac biomarkers were obtained, or before cardiac biomarker values would be increased
• Percutaneous coronary intervention (PCI)-related MI is arbitrarily defined by elevation of cTn values (>5 × 99th percentile URL) in patients with
normal baseline values (≤99th percentile URL) or a rise of cTn values >20% if the baseline values are elevated and are stable or falling. In addition, i)
symptoms suggestive of myocardial ischemia, ii) new ischemic ECG changes, iii) angiographic findings consistent with a procedural complication,
or iv) imaging demonstration of new loss of viable myocardium or new regional wall motion abnormality are required
• Stent thrombosis associated with MI when detected by coronary angiography or autopsy in the setting of myocardial ischemia and with a rise
and/or fall of cardiac biomarker values with at least 1 value above the 99th percentile URL
• Coronary artery bypass grafting (CABG)-related MI is arbitrarily defined by elevation of cardiac biomarker values (>10 × 99th percentile URL) in patients
with normal baseline cTn values (≤99th percentile URL). In addition, i) new pathological Q waves or new LBBB, ii) angiographically documented new graft
or new native coronary artery occlusion, or iii) imaging evidence of new low of viable myocardium or new regional wall motion abnormality are required
TABLE 9–2 Diagnoses of Increased Cardiac Troponin: Elevation of Cardiac

Troponin Values Because of Myocardial Injury
Injury related to primary myocardial ischemia
Plaque rupture
Intraluminal coronary artery thrombus formation
Injury related to supply/demand imbalance of myocardial ischemia

Tachyarrhythmias/bradyarrhythmias
Aortic dissection or severe aortic valve disease
Hypertrophic cardiomyopathy
Cardiogenic, hypovolemic, or septic shock
Severe respiratory failure
Severe anemia
Hypertension with or without LVH
Coronary spasm
Coronary embolism or vasculitis
Coronary endothelial dysfunction without significant CAD
Injury not related to myocardial ischemia

Cardiac contusion, surgery, ablation, pacing, or defibrillator shocks
Rhabdomyolysis with cardiac involvement
Myocarditis
Cardiotoxic agents, for example, anthracyclines, Herceptin
Multifactorial or indeterminate myocardial injury

Heart failure
Stress (takotsubo) cardiomyopathy
Severe pulmonary embolism or pulmonary hypertension
Sepsis and critically ill patients
Renal failure
Severe acute neurological diseases, for example, stroke, subarachnoid hemorrhage
Infiltrative diseases, for example, amyloidosis, sarcoidosis
Strenuous exercise
TABLE 9–3 Universal Classification of Different Types of Myocardial Infarction

Type 1: spontaneous myocardial infarction
Spontaneous myocardial infarction related to atherosclerotic plaque rupture, ulceration, assuring erosion, or dissection with resulting intraluminal
thrombus in 1 or more of the coronary arteries leading to decreased myocardial blood flow or distal platelet emboli with ensuing myocyte necrosis.
The patient may have underlying severe CAD but on occasion nonobstructive or no CAD. These are either an ST-segment elevation MI (STEMI) or
non-STEMI (NSTEMI)
Type 2: myocardial infarction secondary to an ischemic imbalance

In instances of myocardial injury with necrosis where a condition other than CAD contributes to an imbalance between myocardial oxygen supply
and/or demand, for example, coronary endothelial dysfunction, coronary embolism, tachyarrhythmias/bradyarrhythmias, anemia, respiratory
failure, hypotension, and hypertension with or without LVH
Type 3: myocardial infarction resulting in death when biomarker values are unavailable
Cardiac death with symptoms suggestive of myocardial ischemia and presumed new ischemic ECG changes or new LBBB, but death occurring
before blood samples could be obtained, before cardiac biomarker could rise, or in rare cases cardiac biomarkers were not collected
Type 4a: myocardial infarction related to percutaneous coronary intervention (PCI)

Myocardial infarction associated with PCI is arbitrarily defined by elevation of cTn values 5 × 99th percentile URL in patients with normal baseline
values (<99th percentile URL) or a rise of cTn values >20% if the baseline values are elevated and are stable or falling. In addition, i) symptoms
suggestive of myocardial ischemia, ii) new ischemic ECG changes or new LBBB, iii) angiographic loss of patency of a major coronary artery or side
branch or persistent slow- or no-flow or embolization, or iv) imaging demonstration of new loss of viable myocardium or new regional wall motion
abnormality are required
Type 4b: myocardial infarction related to stent thrombosis

Myocardial infarction associated with stent thrombosis is detected by coronary angiography or autopsy in the setting of myocardial ischemia and
with a rise and/or fall of cardiac biomarkers values with at least 1 value above the 99th percentile URL
Type 5: myocardial infarction related to coronary artery bypass grafting (CABG)

Myocardial infarction associated with CABG is arbitrarily defined by elevation of cardiac biomarker values >10 × 99th percentile URL in patients
with normal baseline cTn values (<99th percentile URL). In addition, i) new pathological Q waves or new LBBB, or new LBBB, ii) angiographically
documented new graft or new native coronary artery occlusion, or iii) imaging evidence of new loss of viable myocardium or new regional wall
motion abnormality are required
Further, the universal classification of different types of myocardial infarction is highlighted

in Table 9-3.
cTn testing is most useful when patients are having nondiagnostic ECG tracings. Patients
with AMI can be categorized into several groups based on time of presentation. First, there is the
group of patients who present very early to the emergency department (ED), within 0 to 4 hours
after the onset of ischemic symptoms that include chest pain, without diagnostic ECG evidence of
AMI. For laboratory tests to be clinically useful, biomarkers (cTn) of MI must be released rapidly
from the heart into the circulation to provide sensitive and tissue-specific diagnostic information.
Further the analytical assays using serum, plasma, or whole blood specimens must be rapid and
sensitive enough to distinguish small changes within the reference interval. The second group
includes those presenting 4 to 48 hours after the onset of ischemic symptoms, without evidence of
AMI on ECG. In this group of patients, the diagnosis of AMI requires serial monitoring of both
cTn and ECG changes. The third group presents more than 48 hours after the onset of symptoms
of ischemia with nonspecific ECG changes. The ideal biomarker, again cTn, of myocardial injury
in this group would persist in the circulation for several days to provide a late diagnostic time
window. The shortfall of such a marker might be its inability to distinguish recurrent injury from
old injury, thus the importance of following a rising or falling pattern. The fourth group includes
those who present to the ED at any time after the onset of ischemic symptoms with clear ECG
evidence of AMI, either a STEMI or Q-wave MI. In this group, detection with biomarkers of
myocardial injury is theoretically not necessary.
Cardiac Troponin
The contractile proteins of the myofibril include the regulatory protein troponin. Troponin
is a complex of 3 protein subunits, troponin C (the calcium-binding component), troponin I
(the inhibitory component), and troponin T (the tropomyosin-binding component) (TIC).
The subunits exist in isoforms distributed between cardiac muscle and slow and fast twitch
skeletal muscle. Troponin is localized primarily in the myofibrils (94%–97%), with a smaller
cytoplasmic fraction (3%–6%). cTn subunits I and T have different amino acid sequences
encoded by different genes allowing for their cardiac tissue specificity. Following myocardial
injury, multiple forms are elaborated both in tissue and in blood. The multiple forms of Troponin is a complex
cTnI include the T–I–C ternary complex, IC binary complex, and free I. Multiple chemical of 3 protein subunits,
modifications of these 3 forms can occur, involving oxidation, reduction, phosphorylation troponin C (the calcium-
and dephosphorylation, and both C- and N-terminal degradation. The conclusions from binding component),
these observations are that cTn immunoassays need to be developed in which the antibodies troponin I (the
recognize epitopes in the stable region of cTnI and, ideally, demonstrate an equimolar response inhibitory component),
to the different cTnI forms that circulate in the blood. and troponin T (the
tropomyosin-binding
component). Over the
Analytical Methods for Measuring Cardiac Troponin past 20 years, numerous
manufacturers have
Over the past 20 years, numerous manufacturers have developed monoclonal antibody-based
developed monoclonal
diagnostic immunoassays for the sensitive measurement of cTnI and cTnT. Assay times range
antibody-based
from 5 to 30 minutes. Table 9–4 shows analytical characteristics of representative assays approved diagnostic immunoassays
by the FDA for patient testing. In clinical practice, 2 obstacles limit the ease for switching from for the measurement of
1 cTnI assay to another. First, there is currently no primary reference cTnI material available for cTnI and cTnT.
manufacturers to use for standardizing assays. Second, concentrations fail to agree because of
the different epitopes recognized by the multiple, different antibodies used in different assays.
Therefore, standardization of cTnI assays remains elusive. For cTnT, there is only 1 manufacturer.
Therefore, there are no standardization problems. In 2012, the IFCC Task Force on Clinical Appli-
cations of Cardiac Biomarkers readdressed quality specification aspects for cTn assays. These
specifications were intended for use by the manufacturers of commercial assays and by clinical
TABLE 9–4 Analytical Characteristics of Representative Contemporary Sensitive,

and High-sensitivity (hs), Point-of-care Cardiac Troponin Assays
Epitopes Recognized by
Company/Platform/ 99th Percentile 10% CV Capture (C) and Detection (D)
Assay LoD (μg/L) (μg/L) (μg/L) Antibodies
Abbott ARCHITECT 0.009 0.028 0.032 C: 87-91, 24-40; D: 41-49
Abbott i-STAT (POC) 0.02 0.08 0.10 C: 41-49, 88-91; D: 28-39, 62-78
Alere Triage (POC) 0.05 <0.05 NA C: NA; D: 27-40
Beckman Access AccuTnI 0.01 0.04 0.06 C: 41-49; D: 24-40
Mitsubishi Pathfast (POC) 0.008 0.029 NA C:41-49; D: 71-116, 163-209
Ortho Vitros ECi ES 0.012 0.034 0.034 C: 24-40, 41-49; D: 87-91

a
Radiometer AQT90 (POC) 0.009 0.023 0.039 C: 41-49, 190-196; D: 137-149
Roche Elecsys 2010 0.01 <0.01 0.030 C: 125-131; D: 136-147
Siemens Centaur Ultra 0.006 0.04 0.03 C: 41-49, 87-91; D: 27-40
Siemens Stratus CS (POC) 0.03 0.07 0.06 C: 27-32; D: 41-56
hs-cTnI ng/L ng/L ng/L

a
Abbott ARCHITECT 1.2 16 (5.6%) 3.0 C: 24-40; D: 41-49
Beckman Access 2-3 8.6 (10%) 8.6 C: 41-49; D: 24-40
Singulex Errena 0.09 10.1 (9.0%) 0.88 C: 41-49; D: 27-41
Siemens VISTA 0.5 9 (5.0%) 3.0 C: 30-35; D: 41-56, 171-190
hs-cTnT
Roche Elecsysa 1.0 13 (8%) 12.0 C: 125-131; D: 136-147
LoD, limit of detection; POC, point of care.
a
Available outside the United States.
Data from Apple FS, Collinson PO; IFCC Task Force on Clinical Applications of Cardiac Biomarkers. Analytical characteristics of high
sensitivity cardiac troponin assays. Clin Chem. 2012:58:54–61.
laboratories using cTn assays to establish uniform criteria so that all assays could be evaluated
objectively for their analytical qualities and clinical performance. Factors addressed included:
antibody selection, calibration materials, imprecision characteristics at clinical decision values,
effects of storage time and temperature, glass versus plastic tubes versus gel separator tubes, the
influence of anticoagulants, and whole blood measurements.
99th Percentile Reference Value as a Cutoff for Diagnosis

of Acute Myocardial Infarction
The Global Task Force’s 2012 “Third Universal Definition of Myocardial Infarction” guideline
was predicated on cTn monitoring, with detection of a rising and/or falling cTn, and with at
least 1 value above the 99th percentile value. Using the 99th percentile value (compared with the
older WHO criteria) has demonstrated an increase in the number of MIs in day-to-day clinical
practice, EDs, epidemiologic studies, and clinical trials. The data suggest that the more analyti-
cally sensitive cTn tests result in greater rates of MI diagnosis and greater rates of cTn positivity
compared with the older biomarker CKMB. Milder and smaller MIs are detected. Clinical cases
prior to 2007 that were earlier classified as unstable angina are given a diagnosis of MI because of
an increased and rising cTn. Further, procedure-related troponin increases (ie, following angio-
plasty) will be labeled MI (Table 9–3). The importance of small troponin increases has been
confirmed by their association with a poor prognosis.
Several markers should Several biomarkers should no longer be used to evaluate cardiac disease. They include
no longer be used to aspartate aminotransaminase (AST), total CK activity, CKMB isoforms, myoglobin, total
evaluate cardiac disease. lactate dehydrogenase (LD), and LD isoenzymes. These markers have poor specificity for the
They include aspartate detection of cardiac injury because of their wide tissue distribution. Further, CKMB is no longer a
aminotransaminase (AST), recommended biomarker, and is suggested for clinical use only when cTn assays are not available.
total CK activity, CKMB CKMB offers no additional diagnostic value to aid in the timing of the onset of myocardial injury,
isoforms, myoglobin, total infarct sizing, or determination of reinfarction. There is no evidence to support dual testing for
lactate dehydrogenase cTn and CKMB.
(LD), and LD isoenzymes.
Role of Cardiac Troponin for Risk Outcomes Assessment

Patients With Ischemia
In the environment of preventive and evidence-based medicine, the use of cTnI or cTnT mea-
sured in patients with ischemia will allow clinicians to use biomarkers as both exclusionary and
prognostic indicators. The results will assist in determining who is more at risk for AMI and
death, and thereby determine who may benefit from early medical or surgical intervention. Such
patients benefit from the use of anticoagulant therapy and the use of platelet antagonists, and an
early invasive strategy. The goal of monitoring cardiac biomarkers in patients suggestive of ACS
with and without AMI would be to effectively identify patients with unstable coronary disease and
triage them to an appropriate therapeutic regimen. Optimal use of this strategy requires at least 2
blood samples for cTn measurement. General population screening of hospitalized patients with
cTnI or cTnT is not recommended at present.
Patients With Nonischemic Presentations

Clinicians are often confronted with a clinical history of a patient without overt CAD and a
low probability of myocardial ischemia. However, as a precautionary measure, serial cTns are
ordered. A typical serial order set to rule in or rule out an AMI would include blood draws at
0 hour (presentation), 3, 6, and 9 to 12 hours. When 1 or 2 of the serial cTn concentrations are
found to be increased, the clinician would likely be confronted with the following concerns:
1) What does the increase mean in the clinical setting of a nonischemic patient? 2) Is the increase
a false-positive finding resulting from an analytical error? 3) Why was the test ordered in the
first place? As cTn assays with increasing low-end analytical sensitivity (high-sensitivity [hs] cTn
assays) have been developed (currently not FDA cleared for use in the United States), the ability
to detect minor degrees of myocardial injury in a variety of clinical conditions has widened and
has led to a better understanding that cTn is not just a biomarker for MI, but a sensitive biomarker
for myocardial injury. The 20% of suspected ACS patients who clinically do not rule in for MI,
but display an increased cTn, represents patients with nonischemic pathologies (Table 9–2) in
whom the mechanisms of injury are well defined (such as myocarditis, blunt chest trauma, and
chemotherapeutic agents), and patients with increased cTn, in whom the mechanism of injury
is not clear.
Orders for Serial Cardiac Troponin Testing

Blood samples should be drawn at presentation (0 hour) to the hospital (often this is hours after
the index clinical symptom onset) and at least once more at 6 to 9 hours later. As noted, a typical
serial order set to rule in or rule out an AMI would include blood draws at 0 hour (presentation),
3, 6, and 9 to 12 hours. Occasionally a patient may require a 12- to 24-hour sample, if the earlier
measurements are normal, but the clinical suspicion of AMI is high. As the cTn concentration
may remain increased 3 to 12 days after an AMI, after 2 positive values with a rising pattern,
it does not appear cost-effective to continually monitor cTn once a diagnosis is established. In
patients where recurrent MI is suspected from clinical signs or symptoms following the initial
MI, an immediate remeasurement at the time of a suspicious new event (0) and 3-, 6-, and 9-hour
serial blood samples are recommended. It is reasonable to suspect recurrent infarction if there is
a >20% increase in the second value as long as it exceeds the 99th percentile.
High-sensitivity Cardiac Troponin Assays

It is important to understand that the term “high sensitivity” (hs) reflects the assay’s character-
istics and does not refer to a difference in the form of cTn being measured. There is a need for
a consensus on defining what nomenclature should be used for an hs assay. Several names have
been used in the literature for these assays, but the term “high sensitivity” has been recommended
by expert opinion. This term, however, begs the question: how does one define an hs assay? In a
scorecard concept (Table 9–5), an assay is designated hs if it meets 2 basic criteria. First, the total
imprecision (CV) at the 99th percentile value should be ≤10%. Second, measurable concentra-
tions below the 99th percentile (the upper limit of normal) should be attainable with an assay at
a concentration value above the assay’s limit of detection for at least 50% of healthy individuals.
None of the current US-marketed assays for both central laboratory and point-of-care testing
meet the 2-fold hs criteria. Concentrations for hs assays are expressed in nanograms per liter
instead of the commonly published units of micrograms per liter.
For deriving normal reference 99th percentile cutoffs for cTn assays, it is recommended
that inclusion criteria be based on data obtained from an interview for a history of medications
and known underlying disease, as well as a blood measurement of a natriuretic peptide (NP;
N-terminal pro–B-type natriuretic peptide [NT-proBNP] or B-type natriuretic peptide [BNP]),
interpreted vis-à-vis a cutoff value for the exclusion of ventricular dysfunction to serve as a
TABLE 9–5 Scorecard Designations of cTn Assays

Acceptance Designation Total Imprecision at the 99th Percentile (CV%)
Guideline acceptable ≤10

Clinically usable >10 to ≤20
Not acceptable >20
Assay Designation Measurable Normal Values Below the 99th Percentile (%)
Level 4 (third generation, hs) ≥95
Level 3 (second generation, hs) 75 to <95
Level 3 (first generation, hs) 50 to <75
Level 1 (contemporary) <50

Reproduced with permission from Apple FS. A new season for cardiac troponin assays: it’s time to keep a scorecard. Clin Chem.
2009;55:1303–1306.
surrogate biomarker for underlying myocardial dysfunction, and an estimated GFR to exclude
renal disease. In addition, the groups should be split equally by sex and include a diverse racial
and ethnic mix. Literature now supports reporting of gender-specific cutoffs for hs assays, as men
demonstrate approximately 2-fold higher 99th percentiles compared with women.
With improved analytical sensitivity, hs assays have been shown to provide an earlier diag-
nosis, with the ability to rule in and rule out by 3 hours instead of 6 hours with contemporary
assays. However, with increased clinical sensitivity, with the ability to detect smaller myocardial
injuries from multiple, pathological etiologies, decreased clinical specificity, below 80%, occurs.
Early studies have now demonstrated that the use of a delta change in cTn concentration over a
0- to 3-hour serial time window allows for the ability to separate acute injury, that is, AMI, from
a chronic injury, such as heart failure, with improved clinical specificity up to 95%. This will be
an important tool to use for clinical care when hs assays are cleared for use in the United States
some time in 2014.
CONGESTIVE HEART FAILURE

Description
CHF is a condition in which there is ineffective pumping of the heart leading to an accumulation
of fluid in the lungs. Typically, it results from a loss of cardiac tissue and subsequent function.
It is defined as the pathophysiological condition in which an abnormality of cardiac function
CHF is a condition is responsible for the failure of the heart to pump sufficient blood to satisfy the requirements
in which there is of the metabolizing tissues. In the United States, CHF is the only cardiovascular disease with
ineffective pumping of an increasing incidence. The National Heart, Lung, and Blood Institute estimates that current
the heart leading to an prevalence is about 5 million Americans with CHF, with an incidence of approximately 400,000
accumulation of fluid in new cases each year. CHF is the leading cause of hospitalization in individuals 65 years and older.
the lungs. Two biomarkers Current prognosis is dependent on disease severity, but overall it is poor. The 5-year mortality
have been well studied is approximately 10% in mild CHF, 20% to 30% in moderate CHF, and up to 80% in end-stage
to assist in these
disease.
clinical settings: B-type
natriuretic peptide (BNP,
pharmacologically active Diagnosis
hormone) and N-terminal
proBNP (NT-proBNP, not Natriuretic Peptides in Monitoring CHF
pharmacologically active Two biomarkers have been well studied to assist in these clinical settings: BNP (pharmacologically
peptide). active hormone) and NT-proBNP (not pharmacologically active peptide). Both blood peptides
are derived from cleavage of the myocardial proBNP peptide following myocardial stress and/or
fluid overload. In general, the clinical evidence for utilization of either biomarker is very similar,
but each has subtle analytical and physiological differences, depending on the pathophysiology
of an individual patient. In the course of this chapter, both NPs are used interchangeably unless
specifically noted.
The ACC/AHA practice guidelines for the evaluation and management of CHF indicate that
the role of NP in the identification of CHF patients remains to be clarified. In contrast, the ESC
has incorporated monitoring NPs into their practice algorithm at the time of patient presentation
alongside the clinical history, physical examination, ECG, and chest x-ray. An abnormal NP find-
ing would trigger an echocardiogram or other imaging modality. NP concentrations in patients
diagnosed with CHF are substantially increased (>1000 ng/L for BNP or >1800 NT-proBNP)
when compared with patients who have minor increases (<300 ng/L) because of left ventricular
(LV) dysfunction without acute CHF. CHF is more common in patients with advanced chronic
renal disease. BNP and NT-proBNP are secreted in a pulsatile fashion from cardiac ventricles
with an approximate half-life for BNP of 22 minutes in blood, with the NT-proBNP half-life on
the order of 2 hours. While 1 mechanism of BNP clearance involves the renal parenchyma, the
kidney is not thought to be the primary site for BNP clearance. The kidney more specifically
affects NT-proBNP clearance. Thus, increases in BNP in hemodialysis patients are thought to
represent both regulatory responses from the cardiac ventricle, resulting from increased wall ten-
sion, and a lack of renal clearance.
Importantly, NPs are not 100% specific for CHF. Increases have been described for other
non-CHF etiologies involving filling pressure defects, including LV hypertrophy, inflammatory
cardiac diseases, systemic arterial hypertension, pulmonary hypertension, acute and chronic
renal failure, liver cirrhosis, and several endocrine disorders (eg, hyperaldosteronism and Cush-
ing syndrome). In CHF patients presenting to the ED, patients admitted tend to have higher BNP
concentrations (>500 ng/L) versus those who are discharged (mean <300 ng/L) at triage. Linear
relationships with increasing BNP/NT-proBNP levels and the severity of CHF (NYHA classifica-
tion I-IV) have been described. The largest prospective trial to date to evaluate the diagnostic
value of BNP is “The Breathing Not Properly Multicenter Study,” from which the level of BNP was
found to be an independent predictor of CHF. Using a blood BNP cutoff concentration of 100 ng/L
for CHF, there was a 90% clinical sensitivity and 75% clinical specificity, with an 81% accuracy.
Without BNP monitoring, clinical judgment and traditional diagnostic methods demonstrated a
diagnostic accuracy of only 74%. The knowledge of BNP reduced the proportion of patients in
whom the clinician was uncertain of the diagnosis from 43% to 11%. Plasma BNP monitoring in
the ED improved the treatment and evaluation of patients with early dyspnea, reducing the time
to discharge and total cost of treatment. Similar data have been shown for the alternate biomarker
NT-proBNP. After an AMI, NP increases in proportion to the size of the infarction, prompting
investigators to explore the role of screening BNP for detection of LV dysfunction. In post-MI
patients, BNP concentrations are inversely correlated with LV ejection fraction. However, there
is inconclusive evidence for the role of BNP screening for asymptomatic LV dysfunction in the Blood NP monitoring
general population. In general, there does not appear to be a distinct advantage to use 1 biomarker can be valuable in the
(BNP or NT-proBNP) over the other in clinical practice. diagnostic setting. In the
Blood NP monitoring can be valuable in the diagnostic setting, where it will possibly improve presence of a normal BNP
the performance of nonspecialist clinicians in diagnosing CHF. In clinical practice, NP monitor- or NT-proBNP, a diagnosis
ing can best be used as a “rule-out” test for suspected cases of new CHF. It is not a stand-alone test of CHF is highly unlikely.
and should not be a replacement for a full clinical assessment, including an echocardiogram when
indicated. In the presence of a normal BNP or NT-proBNP, a diagnosis of CHF is highly unlikely
if concentrations are <100 ng/L for BNP or <300 ng/L for NT-proBNP. Monitoring NP may be
useful in 1) guiding therapy, 2) monitoring the course of the disease, and 3) providing useful risk
stratification information. NPs have been shown to be an independent predictor of cardiovascular
mortality in patients with both CHF and ACS over a 1-year period. Further, BNP or NT-proBNP
monitoring may assist in identifying patients with a lower risk of readmission within the next
30 days before discharge.
Analytical Methods for Measuring Natriuretic Peptides

Table 9–6 shows the current FDA-approved assays for BNP or NT-proBNP. The commercial
assays differ in standardization of measurements and antibodies used in the assay. Assays that
use an antibody that recognizes the N-terminus labile region of BNP (eg, Biosite, Beckman, and
Abbott) demonstrate less analyte stability at room temperature (24 hours) than assays that use
1 of their antibodies recognizing the C-terminus (eg, Siemens [Bayer]). The Roche NT-proBNP
antibody configuration allows for 72 hours of sample stability at room temperature.
TABLE 9–6 Representative Commercial BNP and NT-proBNP Assays

BNP
1. Abbott ARCHITECT and AxSYM
2. Alere Biosite Triage
3. Beckman Coulter Access
4. Siemens (Bayer) Centaur
NT-proBNP
1. Roche Elecsys Cobas
2. Seimens (Dade Behring) Centaur and Vista
3. Ortho-Clinical Diagnostics Vitros
4. Response Biomedical Ramp
5. Mitsubishi Pathfast
Reference Intervals: Medical Decision Cutoff Values

A number of clinical factors affect the BNP and NT-proBNP concentrations, most importantly
age, gender, obesity, and renal function. Significant differences are observed between men
and women (higher), and there are increasing concentrations with age by decade. For BNP
and NT-proBNP, the significance of the results for these assays in relation to the degree of
left ventricle dysfunction remains a debate. For both analytes, there is an inverse relationship
between values and body mass index. For NT-proBNP, establishing reference intervals has
been challenging. Review of both the FDA-approved US package insert and the European assay
package insert reveals substantial differences in what concentrations are considered normal
by age and sex. For BNP, a cutoff of 100 ng/L has been endorsed as demonstrating optimal
sensitivity and specificity. For NT-proBNP, the FDA-approved package insert describes a 2-tier
cutoff by age at <75 years: 125 ng/L and >75 years: 450 ng/L. However, more evidence-based
cutoffs have been derived from the PRIDE/ICON studies based on age and renal function, and
are recommended as follows—age <50 years: >450 ng/L; age ≥50 years: >900 ng/L; all ages: best
negative predictive value <300 ng/L; age <50 years and eGFR >60 mL/min: 450 pg/mL, and
eGFR ≤60 mL/min: 1800 ng/L; age ≥50 years and eGFR >60 mL/min: 900 pg/mL, and eGFR
≤60 mL/min: 1800 ng/L.
Implications for Therapy Using Test Results

for Natriuretic Peptides
The utility of serial measurements of NPs in guiding therapy for chronic heart failure has been
the subject of numerous randomized controlled trials reported in the literature since 2000. The
existing trial data suggest that adjustment of treatment in chronic heart failure according to NP
measurements, used in conjunction with established clinical treatments, is likely to reduce car-
diac mortality and hospital admissions with heart failure, at least in patients with systolic heart
failure who are younger than 75 years and relatively free of comorbidities.
Biological Variability
As BNP and NT-proBNP become more widely used to monitor CHF patients following therapy,
questions have addressed the usefulness of serial monitoring in assisting the success of drug ther-
apy. In a study of 11 patients with CHF, the biological variation for BNP and NT-proBNP was
evaluated using 4 different assays. The findings indicated that a change of 130% for BNP and 90%
for NT-proBNP is necessary before results of serially collected data can be considered clinically
and statistically significant. For example, these findings imply that a decrease from approximately
500 to 250 ng/L would be necessary for a clinician to conclude that therapy was successful in
improving CHF features. Clinicians without this knowledge may inappropriately assume that a
decrease from an admission BNP value of 500 ng/L to a 24-hour post-admission value of 400 ng/L
may have been a result of successful patient management. It has been suggested that following the
admission BNP value, a second BNP value be obtained within 24 hours of discharge to optimize
the diagnostic utility of BNP in the overall assessment of patients with CHF.
Novel Biomarkers for Heart Failure Risk Assessment

In addition to the advances in the understanding of established NP biomarkers in HF, there is
an increased study of the elucidation of novel biomarkers potentially useful for the evaluation
and management of patients with HF, and the growing understanding of important and relevant
comorbidities in HF. Literature on candidate biomarkers from a number of classes will be growing
over the next several years and include: a) myocyte stretch (with assays for ST2, GDF-15), b)
myocyte necrosis (with hs-cTn assays), c) systemic inflammation (with assays for LP-PLA2), d)
oxidative stress (with assays for MPO), e) extracellular matrix turnover (with assays for collagen
propeptides), f) neurohormones (with assays for chromogranin A), and g) biomarkers of
extracardiac processes, such as renal function (with assays for NGAL).
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C H A P T E R
Diseases of Red Blood Cells

Daniel D. Mais 10
LEARNING OBJECTIVES
1. Learn the different causes of anemia and their pathophysiology.
2. Learn how to identify the specific cause of anemia in a particular patient.
3. Learn the causes of erythrocytosis and how to distinguish among them.
CHAPTER OUTLINE
Anemia 221 Glucose-6-phosphate Dehydrogenase
Definition 221 (G6PD) Deficiency 246
Differential Diagnosis 222 Pyruvate Kinase (PK) Deficiency 246
Acute Blood Loss 235 Paroxysmal Nocturnal Hemoglobinuria 247
Iron Deficiency Anemia 236 Sideroblastic Anemia 247
Anemia of Chronic Disease (ACD) 237 Pure Red Cell Aplasia 247
Thalassemia 238 Erythrocytosis 248
Folate Deficiency 239 Definition 248
Vitamin B12 Deficiency 239 Differential Diagnosis 248
Lead Poisoning (Plumbism) 240 Polycythemia Vera 248
Sickle Cell Anemia and Other Methods 249
Hemoglobinopathies 241 Red Cell Indices 249
Hereditary Spherocytosis 242 Reticulocyte Counting 250
Hereditary Elliptocytosis (HE) 243 Hemoglobin Electrophoresis 250
Autoimmune Hemolytic Anemia 243 Screening Tests for Sickle Hemoglobin 251
Hemolytic Disease of Osmotic Fragility Test 251
the Newborn (HDN) 245 Direct Antiglobulin Test (Coombs Test) 251
Microangiopathic Hemolytic Anemias 245 Kleihauer–Betke Test 251
ANEMIA
Definition
Anemia refers to a deficiency in red blood cells (RBCs) and implies a decline in oxygen-carrying
capacity. The complete blood count (CBC) provides several measures of red cell quantity, includ-
ing RBC count, hemoglobin (Hb) concentration, and hematocrit (Hct) (see description of RBC
indices later in this chapter). Hb concentration is the parameter most widely used to diagnose
anemia, based on 1967 World Health Organization (WHO) recommendations (Table 10–1). This
definition is not universally accepted, and numerous alternatives have been proposed over the
years, usually suggesting slightly higher values and race-specific values. It is important to remem-
ber also that the normal ranges for Hb and Hct are different for infants, children, adult men, Anemia refers to a
adult women, pregnant women, and the elderly. Attention to age- and gender-appropriate normal deficiency in red blood
ranges is important in the evaluation of anemia. cells (RBCs) and implies
Anemia may present with pallor, fatigue, dyspnea, or evidence of poor tissue oxygen- a decline in oxygen-
ation (chest pain due to poor cardiac oxygenation, altered mental status due to poor cerebral carrying capacity.
221
222 CHAPTER 10 Diseases of Red Blood Cells
TABLE 10–1 WHO Definition of Anemia

Group Hemoglobin (g/dL)
Infants and children, 6 months to 6 years <11.0
Pregnant females <11.0
Children, 6-14 years <12.0
Nonpregnant adult females <12.0
Adult males <13.0
oxygenation). Often, particularly when anemia is mild or the patient is otherwise healthy, anemia
presents simply as an abnormal CBC.
Anemia stimulates several compensatory mechanisms. The cardiopulmonary system com-
pensates by attempting to make the most of the blood it has by exchanging more gases (tachypnea),
and circulating more volume (tachycardia). The marrow responds with increased erythropoiesis,
stimulated by an increase in renal production of erythropoietin (EPO) in response to hypoxia.
If the means to create mature red cells are intact (ie, if the underlying cause of the anemia is not
a production or maturation defect), then this response can usually succeed. In addition to mak-
ing more erythrocytes, the marrow begins to release immature erythrocytes into the circulation.
Many of these still contain a network of ribosomes and rough endoplasmic reticulum involved
in the making of Hb, which identifies them morphologically as reticulocytes (see description of
reticulocyte counting later in this chapter). Over the next 3 to 4 days, this endoplasmic reticu-
Identifying the cause lum dissolves and a mature RBC results. In very brisk marrow responses, some red cells may be
of anemia is usually released that still contain a nucleus.
fairly straightforward.
Examination of the
peripheral smear is Differential Diagnosis
especially important, since Identifying the cause of anemia is usually fairly straightforward. There are several strategies
numerous clues can be for reaching the diagnosis (Tables 10–2 and 10–3), 1 of which is illustrated in the algorithms
found there. (Figures 10–1 to 10–4). Examination of the peripheral smear is especially important, since
numerous clues can be found there (Table 10–4). Figures 10–5 to 10–24 show many of the abnor-
mal morphologies and intracellular inclusions of RBCs.
The reticulocyte count is an important piece of information. When markedly elevated,
this is usually noticeable in a Wright-stained peripheral blood smear. Reticulocytes appear as
large, polychromatophilic red cells, and when they are numerous, the smear is often described
as having polychromasia. Anemia due to a production defect is associated with a normal
TABLE 10–2 Classification of Anemia by Pathophysiology

Production Defect Survival Defect
Proliferation defect Hemolysis

Anemia of chronic disease Hemoglobinopathies
Renal disease (low erythropoietin states) Immune hemolytic anemias
Fanconi anemia Infectious causes of hemolysis
Blackfan–Diamond syndrome Membrane abnormalities
Parvovirus infection Metabolic abnormalities
Drugs or toxins Mechanical hemolysis
Drugs or toxins
Wilson disease
Maturation defect Hemorrhage

Vitamin B12 deficiency Hypersplenism
Folate deficiency
Iron deficiency
Sideroblastic anemia
Lead poisoning
CHAPTER 10 Diseases of Red Blood Cells 223
TABLE 10–3 Classification of Anemia by Mean Corpuscular Volume (MCV) and Red
Blood Cell Distribution Width (RDW)
Normal RDW High RDW
Low MCV Anemia of chronic disease Iron deficiency anemia

Thalassemia Sickle cell disease
Hemoglobin E
Normal MCV Acute blood loss Early nutritional (iron, B12, folate) deficiency
Anemia of chronic disease Sickle cell disease
Low erythropoietin states (renal failure)
High MCV Aplastic anemia Folate and B12 deficiency

Liver disease Myelodysplasia
Alcohol abuse Reticulocytosis (eg, hemolysis)
Microcytic anemia
Reticulocyte count
Hypo-regenerative Hyper-regenerative
microcytic anemia microcytic anemia
Serum ferritin Spherocytosis

Serum iron
% Transferrin saturation
Yes No
Test results Test results

are low are normal Direct Inherited
antiglobulin membrane
test or enzyme
defects
Probable Hemoglobin
iron deficiency electrophoresis
Positive Negative
Sideroblastic anemia Immune

Thalassemia Hereditary
Anemia of hemoglobin E hemolytic spherocytosis
chronic disease anemia
FIGURE 10–1 Diagnostic algorithm for microcytic anemia.

Normocytic anemia
Reticulocyte Multiple cytopenias: Reticulocyte count

count normal Myelodysplasia increased
Bone marrow infiltration
Aplastic anemia
Isolated anemia: No blood loss:

Anemia of chronic disease Probable hemolysis
Early iron deficiency
Early folate/B12 deficiency
Medication effect
If all excluded:
Pure red cell aplasia
Myelodysplasia
Paroxysmal nocturnal
Hemoglobinuria
FIGURE 10–2 Diagnostic algorithm for normocytic anemia.
Macrocytic anemia
Reticulocyte count low Reticulocyte count high
No hypersegmented Macrocytosis due to

Hypersegmented neutrophils and reticulocytosis
neutrophils and MCV < 115: (anemia probably due
MCV > 115: Liver disease to hemolysis or blood
Probable B12 Hypothyroidism loss) or partially treated
or folate deficiency Down syndrome hypo-regenerative
Medication effect macrocytic anemia
Low B12 and All excluded

Normal B12
folate confirms
and folate
deficiency
Probable
myelodysplasia
FIGURE 10–3 Diagnostic algorithm for macrocytic anemia.

Suspected hemolytic anemia

(reticulocytosis, increased LDH):
Exclude splenic enlargement
and occult bleed
Perform:
Peripheral smear
Bilirubin
Haptoglobin
Urine hemoglobin
Urine hemosiderin
Urine urobilinogen
Schistocytes Spherocytes, spur cells, bite cells, etc.

Bilirubin normal to increased Bilirubin increased (especially indirect)
Haptoglobin decreased or absent Haptoglobin normal to decreased
Urine hemoglobin positive Urine hemoglobin absent
Hemosiderinuria present Hemosiderinuria absent
Urobilinogen absent Urobilinogen increased
Intravascular hemolysis Extravascular hemolysis
Microangiopathic: DIC, TTP, HUS, Membrane defects: eg, HS, HE

HELLP (clinical presentation) (peripheral smear)
Mechanical hemolysis, eg, heart Pyruvate kinase deficiency (PK)
valve (note history) (peripheral smear, PK assay)
Toxins, eg, venoms (note history) Hemoglobinopathy (hemoglobin
Infections, eg, malaria, babesia, electrophoresis)
clostridium (peripheral smear, history) Thalassemia (red cell indices,
Oxidant stress, eg, some cases of hemoglobin electrophoresis)
G6PD deficiency (G6PD assay) Hemolytic transfusion reaction:
Hemolytic transfusion reaction, eg, eg, Rh, Duffy (history)
ABO incompatibility (history) Oxidant stress, eg, some cases of
Paroxysmal nocturnal hemoglobinuria G6PD deficiency (G6PD assay)
(Ham’s test, flow cytometry)
Paroxysmal cold hemoglobinuria
(detect anti-P antibody)
FIGURE 10–4 Diagnostic algorithm for suspected hemolytic anemia. DIC, disseminated
intravascular coagulation; TTP, thrombotic thrombocytopenic purpura; HUS, hemolytic uremic
syndrome; HELLP, hemolysis, elevated liver function tests, and low platelets; HS, hereditary
spherocytosis; HE, hereditary elliptocytosis; G6PD, glucose-6-phosphate dehydrogenase.
TABLE 10–4 Morphologic Findings in Red Cells

Finding Definition Associated Conditions
Basophilic stippling Small blue dots in red cells, due to clusters of Hemolytic anemias
ribosomes Lead poisoning
Thalassemia
Pappenheimer Larger, more irregular, and grayer than Asplenia
bodies basophilic stippling, due to iron-containing Sideroblastic anemia
mitochondria
Heinz bodies Heinz bodies: gray-black round inclusions, Oxidative injury as found in
Bite cells seen only with supravital stains (crystal G6PD deficiency or with unstable
violet). Bite cells: sharp bite-like defects in red hemoglobins
cells where a Heinz body has been removed
in the spleen. Both are due to denatured
hemoglobin
Howell–Jolly bodies Howell–Jolly body: dot-like, dark purple Asplenia

Cabot rings inclusion. Cabot ring: ring-shaped dark purple
inclusion. Both represent a residual nuclear
fragment
Target cells Red cells with a dark circle within the Thalassemia
central area of pallor, reflecting redundant Hemoglobin C
membrane Liver disease
Schistocytes Fragmented red blood cells, with forms such Microangiopathic hemolytic
as helmet-shaped cells, due to mechanical red anemias (MHA): DIC, TTP, HUS, HELLP.
cell fragmentation Mechanical heart valves
Dacrocytes Teardrop or pear-shaped erythrocytes Can be seen in thalassemia and

(teardrop cells) megaloblastic anemia
Often seen in myelophthisis
Echinocytes Red blood cells that have circumferential Uremia

(burr cells) undulations or spiny projections with Gastric cancer
pointed tips Pyruvate kinase deficiency
Acanthocytes Red blood cells that have circumferential Liver disease

(spur cells) blunt and spiny projections with bulbous tips Abetalipoproteinemia
McLeod phenotype
Spherocytes Red cells without central pallor due to Immune hemolytic anemia
decreased red cell membrane Hereditary spherocytosis
Elliptocytes Red cells twice as long as they are wide Iron deficiency
Hereditary elliptocytosis
Stomatocytes Red cells whose area of central pallor is Alcohol abuse

elongated in a mouth-like shape Dilantin exposure
Rh null phenotype (absence of
Rh antigens)
Hereditary stomatocytosis
DIC, disseminated intravascular coagulation; TTP, thrombotic thrombocytopenic purpura; HUS, hemolytic uremic
syndrome; HELLP, hemolysis, elevated liver function tests, and low platelets.
See Figures 10–5 to 10–18 for peripheral smears with abnormal red blood cell morphology.
to low reticulocyte count. Such hyporegenerative anemias include iron deficiency anemia,
anemia of chronic disease (ACD), lead poisoning, folate deficiency, B12 deficiency, myelodys-
plastic syndrome, aplastic anemia, and pure red cell aplasia.
Regardless of the morphology or red cell size, anemia that is accompanied by reticulocytosis
suggests either hemolysis or hemorrhage. Some exceptions should be noted. One is a partially
treated production defect, such as in the early treatment of iron, folate, or B12 deficiency, in which
one may find persistent anemia with reticulocytosis. Second, both hemolytic and blood-loss ane-
mia may eventually lead to depletion of iron, folate, or B12, and they can present as a produc-
tion defect. Lastly, paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia that may
transform to aplastic anemia.
FIGURE 10–5 Peripheral blood smear with acanthocytes. FIGURE 10–6 Peripheral blood smear with the
basophilic stippling.
FIGURE 10–7 Peripheral blood smear with dacrocytes. FIGURE 10–8 Peripheral blood smear with echinocyte.
Hemolytic anemias are those in which red cell survival, normally 120 days, is shortened.
The premature destruction of erythrocytes may occur within the bloodstream (intravascular
hemolysis) or within the reticuloendothelial system (eg, extravascular hemolysis). Intravas-
cular hemolysis is caused by mechanical red cell trauma (microangiopathic hemolytic anemia
[MHA] from mechanical heart valve), complement fixation on the red cell surface (eg, ABO
incompatibility), paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal cold hemoglobin-
uria (PCH), snake envenomation, and infectious agents (eg, malaria, babesiosis, Clostridium).
Extravascular hemolysis is much more common and is typical for all remaining causes of hemo-
lysis. The causes of hemolysis may be inherited or acquired. Inherited forms of hemolytic anemia
usually, but not always, present in early childhood (Table 10–5).
FIGURE 10–9 Peripheral blood smear from a patient with hemoglobin C disease.
FIGURE 10–10 Peripheral blood smear showing a Howell–Jolly body.

FIGURE 10–11 Peripheral blood smear from a patient with iron deficiency, showing hypochromic
and microcytic red cells (arrow) and elliptocytes (arrowhead).
FIGURE 10–12 Slide showing the results of a Kleihauer–Betke test.

FIGURE 10–13 Peripheral blood

smear from a patient with megaloblastic
anemia and hypersegmented
neutrophils.
FIGURE 10–14 Peripheral blood

smear from a patient with megaloblastic
anemia and macroovalocytes.
FIGURE 10–15 Peripheral blood smear from a FIGURE 10–16 A peripheral blood smear stained with
patient with large numbers of elliptocytes. Wright stain showing reticulocytes.
FIGURE 10–17 A peripheral blood smear showing FIGURE 10–18 A peripheral blood smear from a patient
circulating nucleated red blood cells (arrowheads), as with stomatocytes.
well as Howell–Jolly bodies (arrows).
FIGURE 10–19 Peripheral blood smear with sickle cells.
FIGURE 10–20 Peripheral blood smear with schistocytes.

FIGURE 10–21 Peripheral blood smear with spherocytes.
FIGURE 10–22 Peripheral blood smear with target cells.

FIGURE 10–23 Peripheral blood smear from a patient with thalassemia, showing microcytic red
cells, target cells (arrow), and basophilic stippling (arrowhead).
FIGURE 10–24 Peripheral blood smear showing Pappenheimer bodies (arrows). Sometimes
mistaken for Howell–Jolly bodies (Figure 10–10).
TABLE 10–5 Laboratory Distinction of Intravascular and Extravascular Hemolysis

Intravascular Hemolysis Extravascular Hemolysis
Schistocytes Microspherocytes
↑ Lactate dehydrogenase (LD) ↑ LD
↓ Haptoglobin Normal to ↓ haptoglobin
↑ Free hemoglobin, ↑ urine hemoglobin ↑ Indirect bilirubin
Hemosiderinuria ↑ Urine and fecal urobilinogen
Hemolytic anemia presents with jaundice, fatigue, tachycardia, and pallor. Enhanced excre-
tion of Hb breakdown products often leads to the development of pigmented gallstones. Intravas-
cular hemolysis may present with dark urine and back pain. Leg ulcers are common in sickle cell
disease and hereditary spherocytosis (HS). Splenomegaly is a common finding in extravascular
hemolysis. Laboratory findings in support of hemolysis include increased unconjugated bilirubin,
increased lactate dehydrogenase (LD), and decreased haptoglobin. Reticulocytes, which are larger
than mature red cells, are responsible for an unpredictability of the mean corpuscular volume
(MCV). The blood smear may display helpful morphologic findings. Intravascular hemolysis is
associated with hemoglobinuria and hemosiderinuria.
Acute Blood Loss

Description
Acute blood loss (hemorrhage) is seen most often as a result of surgery, trauma, or gastrointesti-
nal pathology. Most often, hemorrhage is quite obviously present, but occasionally it is occult and
internal (large retroperitoneal or pelvic hemorrhages). It can occur in the prehospital setting, and Acute blood loss
in that case its volume cannot be estimated. (hemorrhage) is seen
The cardinal manifestations of acute blood loss—tachycardia, tachypnea, and hypotension— most often as a result
reflect not so much a decreased oxygen-carrying capacity as a decreased intravascular volume. of surgery, trauma,
A shift of water from the interstitial fluid compartment into the plasma leads to hemodilution or gastrointestinal
and a lowered hematocrit (hct). It is for this reason that the initial treatment is intravenous fluid pathology.
resuscitation with normal saline; only if this is unsuccessful is blood transfusion considered.
Chronic slow blood loss is generally well tolerated and usually presents late in the disease
process as iron deficiency anemia. Acute blood loss is not the only form of anemia that can present
abruptly. Causes other than hemorrhage that may present as rapid-onset severe anemia include
intravascular hemolysis and acute exacerbations of a chronic compensated hemolytic anemia,
such as in sickle cell disease (Table 10–6).
TABLE 10–6 Nonhemorrhagic Causes of Acute Severe Anemia

Acute Intravascular Hemolysis Acute Exacerbation of Chronic Hemolysis
Microangiopathic hemolytic anemia Parvovirus B19 bone marrow infection (aplastic crisis)
Mechanical hemolysis (eg, heart valve) Splenic sequestration crisis
Toxins (eg, venoms) Hyperhemolytic crisis
Infections (eg, malaria, Clostridium)
Oxidant stress (especially in glucose-6-phosphate

dehydrogenase deficiency)
Hemolytic transfusion reaction (ABO incompatibility)
Paroxysmal nocturnal hemoglobinuria
Paroxysmal cold hemoglobinuria

TABLE 10–7 Causes of Iron Deficiency

Mechanisms Examples
Iron-poor diet Strict vegetarians
Iron malabsorption Celiac sprue

Small bowel resection
Achlorhydria
Hookworm infection
Chronic blood (iron) loss Menses

Colorectal cancer
Idiopathic pulmonary hemosiderosis
Diagnosis
The history and physical examination are the keys to arriving at the correct diagnosis. In
perplexing situations, it may be necessary to exclude hemolysis. The main laboratory find-
ings are a normocytic anemia with a marked reticulocytosis. The peripheral smear may be
notable only for neutrophilia, a result of mobilization of granulocytes from marginal pools
(demargination), which is a physiologic stress response. Somewhat later, there may be reactive
thrombocytosis.
Iron Deficiency Anemia

Description
Within the cytoplasm of the marrow erythroblast, the predominant activity is the production of
Hb molecules into which iron must be incorporated. Iron from the diet is absorbed principally
in the duodenum. It is carried by transferrin to the marrow where it is internalized into erythro-
blasts and incorporated into protoporphyrin to yield heme. Iron not utilized in this way is stored
bound to ferritin. When there is inadequate iron intake or excessive iron loss (Table 10–7), the
ferritin–iron stores of the reticuloendothelial system become progressively depleted. Red cells
are produced that contain an inadequate concentration of Hb, giving rise to the appearance of
small, hypochromic red cells that are poorly equipped for the carriage of oxygen. Fewer mature
red cells are subsequently produced, lowering the Hct (Table 10–8). The clinical manifestations
include those directly attributable to anemia (fatigue, pallor), in addition to pica (a desire to
ingest solids such as rock, dirt, or ice), atrophic glossitis, koilonychias, and esophageal webs.
The coexistence of esophageal webs and iron deficiency has been called Plummer–Vinson syn-
drome. These latter manifestations are not commonly seen and follow prolonged, untreated iron
deficiency.
Iron deficiency is the most common cause of anemia. Worldwide, the most common cause of
iron deficiency is a dietary lack of iron. In the United States, iron intake is not usually problematic,
although supply can lag demand in some populations, such as toddlers and pregnant women.
The finding of iron deficiency produces an obligation to identify and treat the underlying cause.
In American adults, this underlying cause is usually found within the gastrointestinal tract. Iron
The finding of iron deficiency often is the first sign of an occult gastrointestinal malignancy.
deficiency produces an
obligation to identify
and treat the underlying
cause. In American adults, TABLE 10–8 Stages of Iron Deficiency
this underlying cause is Stage Laboratory Findings Clinical Findings
usually found within the
gastrointestinal tract. Iron store depletion ↓ Serum ferritin, ↓ stainable marrow iron None
Iron deficiency often Impaired erythropoiesis All of the above plus ↑ TIBC, ↓ serum iron, and ↑ RDW None
is the first sign of an
Iron deficiency anemia All of the above plus microcytic, hypochromic anemia Fatigue, pallor
occult gastrointestinal
malignancy. TIBC, total iron-binding capacity; RDW, red blood cell distribution width.
Diagnosis
In many cases, the CBC and peripheral blood findings are highly characteristic: low RBC count,
low MCV, low mean corpuscular hemoglobin concentration (MCHC), and high red cell distribu-
tion width (RDW). The platelet count is often elevated. The peripheral blood shows hypochromic,
microcytic red cells with scattered elliptocytes. This is in contrast to the most commonly enter-
tained other diagnostic consideration, thalassemia, in which the RBC count is high, the RDW
tends to be lower, elliptocytes are not seen, and target cells and basophilic stippling are more
frequent.
To confirm the diagnosis of iron deficiency, the best single test is the serum ferritin. A fer-
ritin above 15 μg/L essentially excludes iron deficiency, and the serum ferritin in iron deficiency
is often below 10 μg/L. Lowered ferritin is the earliest finding in iron deficiency and persists
throughout the course of the illness. The diagnostic difficulty with the use of ferritin is that it is
an acute-phase reactant, an analyte that increases in response to inflammation. It may also be
spuriously elevated in hepatic insufficiency, due to impaired clearance. Thus, other assays may
occasionally be needed to make a diagnosis of iron deficiency anemia.
In established iron deficiency, the serum iron is typically low, the total iron-binding capacity
(TIBC) is elevated, and the percent transferrin saturation is low. These findings are somewhat in
contrast to those seen in anemia of chronic disease (see below). Serum soluble transferrin recep-
tor is elevated whenever there are cells depleted of iron; thus, it is elevated in iron deficiency
anemia and in erythroid hyperplasia (hemolytic anemia, polycythemia). Lastly, the zinc proto-
porphyrin (ZPP) and free erythrocyte protoporphyrin (FEP) are elevated in iron deficiency but
also elevated in lead poisoning and anemia of chronic disease. As a last resort, marrow iron stores
can be examined directly under the microscope if an adequate bone marrow aspirate is obtained.
Anemia of Chronic Disease (ACD)

Definition
Sustained systemic inflammation alters iron utilization in the marrow, suppresses hematopoiesis,
and blunts the response of EPO to anemia. This combination of factors leads to a mild, refractory,
hyporegenerative anemia that is usually normocytic and normochromic, but is microcytic in up
to one third of cases. Although iron deficiency is the most common cause of anemia worldwide,
ACD is the most common cause of anemia in both hospitalized and ambulatory hospital patients
in the United States. The vast majority of cases are due to rheumatoid arthritis, collagen vascular
disease such as lupus, chronic infection, and malignancy.
The means by which chronic inflammatory diseases cause anemia are still being elucidated. ACD is the most common
Marrow biopsies in patients with ACD display bountiful iron stores in the face of decreased cause of anemia in
iron uptake by erythroid precursors. Decreased transferrin receptors have been demonstrated both hospitalized
on erythroblasts in ACD. In addition, patients with ACD have decreased production of EPO in and ambulatory
response to anemia. Cytokines, including IFNγ, TNFα, IL-1, and hepcidin, have been shown to hospital patients in
produce the conditions of ACD when injected into laboratory animals. the United States.
Diagnosis
The diagnosis of ACD is made difficult by the presence of numerous comorbid factors, in patients
who, by definition, are ill. In such patients, ACD may be coincident with iron deficiency, folate
deficiency, renal insufficiency, and/or frequent phlebotomy. Furthermore, in up to 30% of those
with iron indices characteristic of ACD, no chronic illness can be identified.
The laboratory diagnosis of ACD depends on demonstrating a hypoproliferative (low reticu-
locyte count) normocytic or microcytic anemia in the presence of characteristic iron studies. The
iron studies should document increased iron stores (normal to high serum ferritin or increased
stainable iron in a bone marrow biopsy) and a low serum iron, low transferrin, and low TIBC.
A normal or elevated ferritin level is crucial for distinguishing ACD from iron deficiency.
However, interpretation of the results for ferritin can be problematic because ferritin is an acute-
phase reactant. Thus, while a low ferritin is essentially diagnostic of iron deficiency, a normal fer-
ritin does not entirely exclude it. In confusing situations, the soluble serum transferrin receptor
assay may be helpful. This analyte is increased in iron deficiency anemia and normal in ACD.
Thalassemia
Description
Mutations in the genes that encode globin chains may result in 2 broad categories of disease.
Some mutations lead to the production of a structurally abnormal globin chain, resulting in a
hemoglobinopathy such as HbS (sickle cell disease and sickle cell trait). Other mutations lead to
reduced production of a structurally normal globin chain, resulting in thalassemia.
Some mutations lead A Hb molecule is composed of 4 polypeptide chains. The major adult Hb, hemoglobin A
to the production of a (HbA), is composed of 2 α chains and 2 β chains. The minor adult Hb (HbA2) is composed of
structurally abnormal 2 α chains and 2 δ chains. The major fetal hemoglobin (HbF) is composed of 2 α chains and
globin chain, resulting in 2 γ chains. The 1 constant feature of all Hbs is the α chain. The α chain genes are located on
a hemoglobinopathy such chromosome 16. Each chromosome 16 contains 2 separate α chain genes, for a total of 4 genes
as HbS (sickle cell disease per normal cell, each transcriptionally active. Thus, to render an individual completely deficient
and sickle cell trait). of α chains, inheritance of 4 mutated genes is required. The β, γ, and δ chain genes are located on
Other mutations lead chromosome 11. Each chromosome 11 contains 1 β, 1 γ, and 1 δ gene. Should a mutation occur in
to reduced production the β chain, there can be a degree of compensation by increasing the production of γ, δ, or both.
of a structurally normal
There is no such substitute for the α chain.
globin chain, resulting in
With decreased α chain production, α-thalassemia arises. Harm comes to the red cell, how-
thalassemia.
ever, not from a deficiency of α chain, but from an excess of non-α chains (eg, β). The excess
chains form precipitates in the cell, leading to ineffective erythropoiesis, microcytosis, and
enhanced splenic red cell destruction. Likewise, decreased β chain production (β-thalassemia)
leads to precipitation of excess α chains and subsequent red cell destruction. Disease severity
reflects the genotype (Table 10–9).
Diagnosis
Since α chains are present in utero, α-thalassemia can be diagnosed at birth. The diagnosis of
β-thalassemia is somewhat delayed, since β chains are not produced to adult levels until 3 to
6 months of age. The CBC is notable for microcytosis, usually in the presence of a normal or
TABLE 10–9 Thalassemia Syndromes

Category Syndrome Genotype Manifestations
αα/αα
Normal Normal None
β/β
α-Thalassemia (silent) αα/α■
None
carrier β/β
αα/■ ■
β/β
α-Thalassemia minor Mild
α-Thalassemia α■/α■
syndromes β/β
α■/■ ■
Hemoglobin H Moderate to severe
β/β
■ ■/■ ■
Hemoglobin Barts Fatal
β/β
αααα
β/β+
β-Thalassemia minor Asymptomatic
αααα
β/βº
β-Thalassemia αααα
syndromes β-Thalassemia β+/β+
Moderate to severe
intermediate αααα
β+/βº
αααα Severe; transfusion
β-Thalassemia minor
βº/βº dependent
Notation: α, normal α gene; ■, severely suppressed α gene; β, normal β gene; β+, moderately suppressed β gene; βº, severely
suppressed β gene.
high RBC count. The peripheral smear often displays target cells and may display basophilic stip-
pling. When there is microcytosis, “thalassemic” indices, and normal iron studies, the diagnosis
of thalassemia is essentially assured. In the case of β-thalassemia, a Hb electrophoresis displays
increased HbA2 and sometimes HbF (see description of Hb electrophoresis later in this chapter
and in Chapter 2). In α-thalassemia (recall that the α chain is needed for all Hb types), the pro-
portion of hemoglobins appears normal. These findings are usually sufficient for the diagnosis.
If further definition is required, molecular genetic testing is available.
Folate Deficiency
Description
Folate and vitamin B12 deficiency (described next) are the classical causes of megaloblastic ane-
mia. The term megaloblastic refers to the appearance of hematopoietic precursor cells in the
marrow. Their nuclei appear abnormally large and immature, resulting from nuclear maturation
that lags behind cytoplasmic maturation. This megaloblastic change affects not only erythro- Folate and vitamin B12
blasts but other rapidly dividing cells as well, including maturing granulocytes, megakaryocytes, deficiency (described
and enterocytes. It results from impairment of DNA synthesis and has more than just morpho- next) are the classical
logic consequences. causes of megaloblastic
Erythropoiesis becomes ineffective, resulting in a hypercellular marrow. Many erythroblasts anemia.
are destroyed while still in the marrow. Thus, megaloblastic anemia is in part a hemolytic anemia;
indeed, intramedullary destruction of maturing erythrocytes leads to increased LDH and biliru-
bin, as one would associate with hemolytic anemia. The red cells that do proceed to maturity are
macrocytic, with the MCV in fully developed megaloblastic anemia exceeding 115 fL.
Folate deficiency does not cause the same neurologic defect that vitamin B12 deficiency
causes. However, supplementation of folate in early pregnancy is known to reduce the incidence
of neural tube defects. No clear mechanism for this effect has been established.
Dietary factors are a major cause of folate deficiency. Folate is found in leafy green vegetables,
fruits, and legumes. Dietary folate is absorbed in the duodenum, and the body stores about a 4- to
5-month supply of it. Thus, within a relatively short time, poor diet, malabsorption, or excessive
utilization can lead to folate deficiency (Table 10–10).
Diagnosis
The blood smear shows the classic features of megaloblastic anemia: marked oval macrocytosis,
hypersegmented neutrophils, and large platelets. The diagnosis can be confirmed by measuring
the serum or RBC folate. However, there are several confounding factors in the use of these tests.
Several balanced meals can quickly normalize the serum folate, but the RBC folate reflects folate
status better over time. Vitamin B12 deficiency can produce a falsely low RBC folate, but it does
not affect the serum folate.
Vitamin B12 Deficiency

Description
Like folate deficiency, vitamin B12 deficiency leads to megaloblastic anemia. The main differ-
ence between the 2 conditions is that B12 deficiency may also produce a degenerative neurologic
syndrome, the manifestations of which are attributable to demyelination of and loss of nerve
fibers within the dorsal columns. The neurologic symptoms include paresthesia, weakness, and
TABLE 10–10 Causes of Folate Deficiency

Inadequate intake Malnutrition, chronic alcoholism
Malabsorption Celiac sprue, small bowel resection
Increased demand Pregnancy, chronic hemolysis
Renal loss Dialysis

TABLE 10–11 Causes of Vitamin B12 Deficiency

Inadequate intake Strict vegetarians
Malabsorption Pernicious anemia, achlorhydria, gastrectomy, ileal disease or resection,

D. latum infestation
Increased demand Pregnancy, chronic hemolysis
Impaired transport Trancobalamin deficiency
an unsteady gait. It is critical to make the diagnosis of B12 deficiency and treat it appropriately,
because these neurologic changes are not reversible.
Malabsorption is the major cause of vitamin B12 deficiency (Table 10–11), most commonly
from pernicious anemia. Pernicious anemia is a deficiency in gastric intrinsic factor (IF) due to
an autoimmune assault on the gastric mucosa. Unlike folate deficiency, B12 deficiency is rarely
due to a poor diet. This is because 1) B12 is abundant in a wide range of dietary sources and 2) the
body stores several years worth of vitamin B12. Dietary deficiency thus requires multiple years of
a highly restrictive vegetarian diet.
Diagnosis
The blood smear shows the classic features of megaloblastic anemia: marked oval macrocytosis,
hypersegmented neutrophils, and large platelets. The diagnosis can be confirmed by measuring
serum B12 levels.
Identifying the cause of the deficiency is the next step in the evaluation. The Schilling test
is designed for this purpose. The patient is given a parenteral dose of unlabeled B12 followed by
an oral dose of radiolabeled vitamin B12. The purpose of the unlabeled dose is to fully saturate
the body with B12 so that the radiolabeled dose will be quickly excreted in the urine. A 24-hour
urine sample is then collected. A low level of urinary radioactivity confirms B12 malabsorption,
but it does not identify the specific gastrointestinal defect. The second part of the Schilling test is
then undertaken. The patient is given another oral dose of radiolabeled B12 in addition to oral IF.
Patients with pernicious anemia will demonstrate enhanced absorption (increased urinary radio-
activity) in this second part of the test. The Schilling test has been largely supplanted by serologic
tests for autoantibodies, including anti-IF and antiparietal antibodies.
Lead Poisoning (Plumbism)

Description
Lead toxicity affects RBCs, renal epithelium, and the nervous system. It generally presents insidi-
ously, with nonspecific features such as abdominal pain and cognitive impairment. However, it
may present abruptly with vomiting, seizures, and altered mental status. In addition, lead poison-
ing may present as a microcytic, hypochromic anemia. Exposure to lead occurs through envi-
ronmental sources, such as lead-based household paint, contaminated soil, lead plumbing, and
manufacturing facilities.
Lead exerts its hematologic effects in 2 ways: inhibition of heme synthesis in the maturing
Lead exerts its erythrocyte and decreased survival of mature erythrocytes. Lead has a strong affinity for certain
hematologic effects in amino acids, particularly the sulfhydryl group of cysteine, and certain organelles, particularly
2 ways: inhibition of mitochondria. Since heme synthesis takes place within mitochondria, and 2 enzymes instrumen-
heme synthesis in the tal in this process, delta-aminolevulinic acid dehydratase (δ-ALA) and ferrochelatase, are rich
maturing erythrocyte in the sulfhydryl groups, this process is exquisitely sensitive to lead. Ferrochelatase catalyzes the
and decreased survival insertion of iron into the protoporphyrin ring. Its inhibition leads to the accumulation of free
of mature erythrocytes. (iron-free) erythrocyte protoporphyrin (FEP), much of which binds nonenzymatically to zinc
to form zinc protoporphyrin (ZPP). Separate from its effects on heme synthesis, lead inhibits
ATPase-driven sodium channels, leading to increased osmotic fragility and hemolysis. Lastly,
lead inhibits the enzyme 5′-nucleotidase, leading to basophilic stippling.
Despite all these vulnerabilities, anemia does not develop until blood lead levels are above
50 μg/dL. A blood lead level >10 μg/dL is considered elevated. Iron deficiency enhances the
effects of lead toxicity in 2 ways. The absence of iron enhances the blockage of the ferrochelatase
step in heme synthesis, and in an effort to absorb more iron, the gastrointestinal absorption of
lead increases.
Diagnosis
Basophilic stippling is noted in the peripheral blood smear and in maturing erythroblasts in the
marrow. The Centers for Disease Control and Prevention has defined lead poisoning as a blood
lead level >10 μg/dL. Elevations in FEP and ZPP do not occur until blood lead levels exceed
35 μg/dL; thus, these assays are not sufficiently sensitive to screen for lead poisoning.
The advantage of FEP measurement, however, is that it can be performed reliably on small
finger- or heel-prick samples. Furthermore, this assay can easily identify patients grossly intoxi-
cated with lead. Elevated FEP and ZPP are not specific for lead poisoning and may also be seen
in iron deficiency.
Sickle Cell Anemia and Other Hemoglobinopathies

Description
A hemoglobinopathy is a structural defect in Hb, usually resulting from a germline single-nucle-
otide point mutation in 1 of the Hb genes. There are examples of postsynthetic modifications in
normally formed Hb, such as carboxyhemoglobin from carbon monoxide poisoning. The com-
mon hemoglobinopathies are listed in Table 10–12. In the United States, HbS is the most com-
mon abnormal Hb, followed by HbC, and HbE. Worldwide, HbS remains most common, but is
followed closely by HbE (which is very common in Southeast Asia), followed by Hbs C, D, and G.
In all, several hundred structurally abnormal Hbs have been described.
Homozygous sickle cell anemia (genotype SS, sickle cell disease) is associated with abnormal
polymerization of Hb in red cells, leading to a cell with an altered shape that is rapidly cleared
from the circulation. Polymerization of HbS is enhanced in hypoxic conditions. While normal In the United States, HbS
red cells have a life span of about 120 days, the red cells in SS have an average life span less than is the most common
30 days. Hb electrophoresis shows that the red cells contain mostly HbS, with small quantities abnormal Hb, followed
of HbF and HbA2. The clinical course in HbSS patients is one of chronic hemolysis punctu- by HbC, and HbE.
ated by a wide range of complicating events (often called crises). Chronic hemolysis leads to a Worldwide, HbS remains
chronic anemia with growth retardation, delayed puberty, impaired exercise tolerance, jaundice, most common, but is
and cholelithiasis (due to the formation of pigmented gallstones). The patients are usually in need followed closely by HbE
of intermittent transfusions. Episodic complications include vaso-occlusive events (eg, stroke, (which is very common in
avascular necrosis of bone, splenic autoinfarction), splenic sequestration crises, aplastic crises Southeast Asia), followed
by Hbs C, D, and G.
(due most often to marrow infection with parvovirus B19), bacterial sepsis, and hyperhemolytic
crises. The risk of bacterial infection is related to an underlying functional asplenia that affects
most sickle cell patients by late childhood. This confers a particular susceptibility to infection by
encapsulated bacterial organisms such as Haemophilus influenzae and Streptococcus pneumoniae.
The most common cause of death in sickle cell disease is infection, followed by stroke and other
thromboembolic events.
TABLE 10–12 Common Hemoglobinopathies

Hemoglobin Gene
Defects Definition
Hemoglobin S Change in sixth amino acid of the β chain from glutamate to valine (β6 glu → val)
Hemoglobin E Change in 26th amino acid of β chain from glutamate to lysine (β26 glu → lys)
Hemoglobin C Change in 6th amino acid of β chain from glutamate to lysine (β6 glu → lys)
Hemoglobin D Change in 121st amino acid of β chain from glutamate to glutamine (β121 glu → gln)
Hemoglobin G Change in 68th amino acid of α chain from asparagine to lysine (α68 asn → lys)
Heterozygotes (genotype SA, sickle cell trait) are essentially asymptomatic and have normal
red cell indices. The presence of sickle Hb can be detected by Hb electrophoresis, where it is
found to represent about 35% to 45% of total Hb. When exposed to hypoxic conditions such as
high altitude, these patients are at risk for splenic infarcts. Interestingly, patients who are double
heterozygotes for HbS and β-thalassemia are more severely affected than heterozygous SA, having
red cells that contain >50% HbS. Conversely, double heterozygotes for S-α-thalassemia manifest
less HbS (<35%) and less severe symptoms.
HbE is relatively benign clinically, in both heterozygous and homozygous forms. Patients
with HbE have red cell indices, however, that closely resemble those of a thalassemic patient
(microcytic with high RBC count). HbE is prevalent in Southeast Asia. Double heterozygotes for
S and E (SE disease) manifest moderate-to-severe hemolysis.
HbC disease (genotype CC) is generally associated with mild hemolysis, and heterozygotes
(CA) are clinically normal. In both, target cells tend to be numerous in the peripheral smear.
Patients who are doubly heterozygous for S and C (SC disease) have manifestations intermediate
between SS and SA. While manifestations are generally milder than SS, there is a greater inci-
dence of avascular necrosis of bone and retinal damage in SC than in SS. The peripheral blood
film shows both sickle cells and target cells.
Hbs D and G are benign Hb variants. They can lead to confusion in interpreting an abnormal
Hb electrophoresis, since they appear in the same location as HbS. However, these patients are
clinically well.
Diagnosis
The identification of variant Hbs is usually performed with Hb electrophoresis. However, many
laboratories now use high-performance liquid chromatography (HPLC). One limitation of both
of these techniques is that several different variants can give similar results, although this is sig-
nificantly less problematic in HPLC. Findings must be correlated with knowledge of the patient’s
clinical status and red cell indices before a definitive diagnosis can be rendered.
There are a number of screening tests for sickle Hb. These are based on the tendency of HbS
to polymerize. A positive sickle screen is not specific for sickle cell disease, however, and can be
present in sickle cell trait, SC disease, and Hb Charlem. Furthermore, a negative screening test does
not entirely exclude HbS, particularly in infants who may still have significant quantities of HbF,
which inhibits polymerization of HbS.
Hereditary Spherocytosis
Description
HS was once known as hereditary hemolytic jaundice. Its cardinal features are chronic hemo-
lysis, jaundice, and splenomegaly. It is a fairly common condition, particularly among people
of Northern European descent, in whom it is the most common inherited red cell disorder. In
the United States the incidence is about 1 in 5000. HS is usually transmitted as an autosomal
dominant trait, but about 25% of affected families display autosomal recessive inheritance. This
HS was once known as
variation derives from the fact that HS can be caused by any 1 of several defects in RBC cytoskel-
hereditary hemolytic
etal proteins, including band 3, protein 4.2, spectrin, and ankyrin. A deficiency in any of these
jaundice. Its cardinal
features are chronic
components leads to cytoskeletal instability. Subsequently, there is loss of the biconcave shape in
hemolysis, jaundice, and favor of the stoichiometrically more attainable sphere.
splenomegaly. It is a The plurality of underlying molecular defects also contributes to clinical heterogeneity, with
fairly common condition, phenotypes ranging from mild to severe. HS may present early as neonatal jaundice, or it may
particularly among people present in late childhood with splenomegaly and mild anemia. While anemia in some cases is
of Northern European quite severe, in most cases the hemolytic anemia is mild and well compensated by the marrow.
descent, in whom it is the Some patients require splenectomy, which usually results in clinical remission. However, splenec-
most common inherited tomy carries with it an increased susceptibility to bacterial sepsis. As HS patients age, they are at
red cell disorder. risk for pigmented gallstones.
Diagnosis
The peripheral blood film shows numerous spherocytes. These appear as red cells that lack central
pallor. Larger polychromatophilic cells are often numerous, reflective of an increased reticulocyte
count. While spherocytes are typically smaller than normal red cells, the MCV may be low, nor-
mal, or high, owing to reticulocytosis. The MCHC is characteristically increased.
When numerous spherocytes are observed on a peripheral blood film, the 2 primary con-
siderations are immune hemolysis and HS. Immune hemolysis can usually be excluded with a
negative direct antiglobulin test (DAT, Coombs test).
The osmotic fragility test can be useful in supporting the diagnosis of HS. However, sphero-
cytes from any cause will result in a positive test.
Hereditary Elliptocytosis (HE)

Description
This autosomal dominant disorder is due to defective tetramerization of cytoskeletal spectrin,
resulting in elliptocytes, also called ovalocytes. There are several clinical variants. The common
type of HE is seen primarily in African Americans and manifests as a mild lifelong hemolytic ane-
mia. Hereditary pyropoikilocytosis is a variant of HE in which RBCs are exquisitely sensitive to
damage from heat. The peripheral blood smear is notable for a profound degree of poikilocytosis
with red cells of every size and shape. This condition is usually most pronounced in infancy and
tends to abate with age, giving way to a phenotype of common HE. A stomatocytic type of HE
exists that is also called Southeast Asian ovalocytosis. This phenotype confers some protection
against infection by P. vivax malaria.
Diagnosis
There is no specific laboratory test for HE. The diagnosis depends on finding elliptocytes in the
peripheral blood. By definition, these cells are twice as long as they are wide, and in HE they com-
prise more than 25% of all red cells. Elliptocytes are not unique to HE and may be seen in iron
deficiency anemia and thalassemia. The proportion of elliptocytes is usually much less than 25%
in these other conditions, and they are easily excluded on other grounds. Once these are ruled
out, the diagnosis is made of HE.
Autoimmune Hemolytic Anemia

Description
When an antibody attaches to a red cell, the consequences depend largely on the nature of the
antibody. Some antibodies are capable of activating complement and producing brisk intravas-
cular hemolysis. Others behave as opsonins, promoting red cell destruction in the spleen. Some
antibodies react only in a cold environment, some only in warmth. Some coat the red cell and do
nothing more.
These disorders present with the typical manifestations of anemia, with variable rates of
onset. Mild splenomegaly is common when hemolysis is extravascular. Dark urine, abdominal or
back pain, and fever may accompany intravascular hemolysis. In severe IgM-induced cold auto-
immune hemolytic anemia (CAIHA), the skin may have a livedo reticularis pattern, and there
may be acrocyanosis on exposure to cold.
Warm autoimmune hemolytic anemia (WAIHA) is mediated by IgG autoantibodies that
optimally bind RBCs at body temperature (37°C). The red cell antigens most commonly the tar-
get in WAIHA are the Rh antigens. IgG molecules must form cross-links to activate complement,
Warm autoimmune
and the target red cell antigens in WAIHA are usually insufficiently dense on the red cell surface to
hemolytic anemia
permit this. A higher-density antigen is involved in a condition known as PCH, described below.
(WAIHA) is mediated by
Thus, IgG antibodies opsonize the red cell in WAIHA, leading to membrane damage mediated by IgG autoantibodies that
splenic macrophages with the formation of small, spherocytic, cells (microspherocytes). In some optimally bind RBCs
cases, there is concomitant immune thrombocytopenia, and this association is known as Evans at body temperature
syndrome. (37°C). CAIHA, also
CAIHA, also called cold agglutinin disease, is mediated by IgM antibodies that bind RBCs called cold agglutinin
at lower temperature ranges. The target antigens are usually the red cell antigens I or i. Those disease, is mediated
binding over a limited thermal amplitude (eg, 0°C-22°C) will obviously not produce clinical by IgM antibodies that
consequences. However, these antibodies may cause difficulty in the laboratory, where studies bind RBCs at lower
are routinely carried out at room temperature, which could be within this thermal amplitude. temperature ranges.
TABLE 10–13 Drug-induced Immune Hemolytic Anemia

Mechanism Drug absorption (hapten) Immune complex AIHA
Type of hemolysis Extravascular Intravascular Extravascular
Implicated drugs Penicillin Quinidine α-Methyldopa

Ampicillin Phenacetin Mefenamic acid
Methicillin Thiazides L-DOPA
Carbenicillin Rifampin Isoniazid
Cephalothin Sulfonamides Procainamide
Hydralazine
Ibuprofen
Antibodies with broader thermal amplitude may bind to red cells in the extremities, where tem-
perature falls a bit below core body temperature, resulting in acrocyanosis. IgM antibodies are
capable of activating complement. Most often, the clinical consequence is a result of opsonization
by C3, leading to extravascular hemolysis similar to that seen in WAIHA. C3-mediated hemolysis
is more of a hepatic process than a splenic one. Sometimes, however, the complete complement
cascade is activated on the cell surface, resulting in intravascular hemolysis.
Both WAIHA and CAIHA are often idiopathic conditions. However, a significant number are
secondary to another underlying condition, including lymphoid neoplasms (eg, chronic lympho-
cytic leukemia), medication use, systemic autoimmune diseases (eg, systemic lupus erythema-
tosus), immunodeficiency (eg, common variable immunodeficiency), and infection (infectious
mononucleosis, HIV, and Mycoplasma pneumoniae).
Paroxysmal cold hemoglobinuria (PCH) is caused by IgG antibodies that are directed at the
red cell P antigen. The antibody responsible for PCH is called the Donath–Landsteiner antibody.
This particular IgG antibody has peculiar tendencies, including the binding of red cells in colder
temperatures (in the blood of the extremities) and the activation of complement, producing intra-
vascular hemolysis. Originally described in association with syphilis, the antibody now is more
often seen in children with viral infections. Mortality can be quite high, up to 30%.
Drug-induced immune hemolytic anemia arises through several pathophysiologic mech-
anisms (Table 10–13). An antibody may be raised against a drug that is capable of adhering
nonspecifically to the red cell membrane (drug adsorption or hapten mechanism). Second, drug–
antibody immune complexes may coat the red cell surface (immune complex mechanism). What
distinguishes these first 2 mechanisms is that the antibody is directed against the drug, not a red
Drug-induced immune cell antigen. Lastly, a drug may be responsible for eliciting a true autoimmune hemolytic anemia,
hemolytic anemia with antibody against red cell antigens. This condition is clinicopathologically indistinguishable
arises through several from AIHA, and it may or may not abate when the drug is discontinued.
pathophysiologic Lastly, alloimmune hemolytic anemia is due to transfusion of red cells bearing an anti-
mechanisms. gen foreign to the recipient. Most responsible antibodies arise as a result of prior sensitization,
commonly prior transfusion or pregnancy, and most cause extravascular hemolysis of mild-to-
moderate severity. In the case of ABO antigens, the antibodies are naturally occurring, and prior
sensitization is not required for there to be a problem. Furthermore, ABO antibodies produce
severe intravascular hemolysis, which can be fatal.
Diagnosis
The DAT, also known as the direct Coombs test, is pivotal for the diagnosis of immune hemolysis.
This test is capable of demonstrating the presence of antibodies or complement on the surface of
RBCs.
Additional laboratory findings include anemia, reticulocytosis, indirect hyperbilirubinemia,
decreased haptoglobin, and an increased LDH. The peripheral blood smear often demonstrates
spherocytes, polychromasia, and, in severe cases, nucleated red cells. In cold agglutinin disease,
red cell clumping is seen.
An important consequence of red cell antibodies is their tendency to interfere with pretrans-
fusion testing.
Hemolytic Disease of the Newborn (HDN)

Description
If there is mingling of fetal and maternal blood (a fetomaternal hemorrhage), then the mother
can become sensitized to antigens of the fetal blood cells. Some of these antigens are paternal in
origin and may therefore be foreign to the mother, and a maternal antibody reaction may occur.
If the antibody idiotype produced is one that can cross the placenta (most IgG subtypes can cross
the placenta, IgM cannot), it can produce fetal hemolysis.
The severity of fetal hemolysis depends on several factors, including the identity of the
immunizing antigen and the titer of maternal antibody. The pregnancy that creates sensiti-
zation is usually spared, as the initial reaction produces largely IgM that does not cross the
placenta. In subsequent pregnancies, an IgG-mediated anamnestic response may be raised,
producing HDN. Furthermore, pregnancy-induced maternal sensitization may complicate
future transfusions.
When this syndrome was first recognized, it was most commonly associated with antibodies
to the Rh antigen known as D. This D antigen is the basis for categorizing blood types as Rh+ or
Rh−. However, prevention strategies have reduced the incidence of RhD HDN to about 0.1% of all
pregnancies. The incidence of maternal anti-Kell antibody now exceeds that of anti-D antibodies
in many centers.
If a pregnant woman does have antibodies against a fetal antigen, the fetus is at risk for
HDN. Mild HDN may only manifest as compensated hemolysis in which fetal erythropoiesis
is capable of keeping up with the rate of red cell destruction. Severe HDN manifests with fetal If a pregnant woman does
anemia, hyperbilirubinemia, and numerous circulating nucleated RBCs (erythroblastosis fetalis). have antibodies against a
Hypoproteinemia may ensue, leading to decreased serum osmotic pressure, and severe edema fetal antigen, the fetus is
(hydrops fetalis). A pregnancy in which there is known sensitization (maternal antibodies to at risk for HDN.
fetal red cell antigens have been detected) must be monitored to determine the severity of fetal
hemolysis.
RhD HDN is prevented by the administration of Rh immune globulin (RhIg) to Rh-negative
women during pregnancy. The RhIg binds to and effectively conceals D antigenic sites, precluding
an immune response. RhIg is given routinely at 28 weeks, at term, and whenever a fetomaternal
hemorrhage is suspected (amniocentesis, trauma, abortion, abruption, etc).
Diagnosis
Several laboratory tests support the diagnosis and treatment of HDN. First, there is blood typing
to confirm the maternal, paternal, and neonatal Rh status.
Second, in Rh-negative women, a screening test for antibodies must be performed. This is a
test in which maternal serum is incubated with a panel of red cells having known antigenic status.
If an alloantibody is detected, its titer is determined by serially diluting the sample until reactivity
is abolished. If a titer of >1:32 is present, the risk of HDN is considered sufficiently high to war-
rant fetal monitoring.
In Rh-negative women with alloantibodies, the fetus must be monitored to determine the
severity of hemolysis. Amniocentesis is performed to determine the quantity of amniotic fluid
bilirubin. When low, monitoring is continued. When high, consideration is given to therapeutic
intervention, including intrauterine transfusion and, when possible, delivery.
In Rh-negative women without antibodies, laboratory tests are available to confirm and
quantify a fetomaternal hemorrhage. These include the Kleihauer–Betke test, the erythrocyte
rosette test, and others. If positive, a dose of RhIg may be given.
Microangiopathic Hemolytic Anemias

Description
This group of disorders shares the ability to create a microvascular environment capable of shred-
ding red cells. They do this usually by inducing endothelial injury and thrombosis, generating a
jagged lattice of fibrin against which red cells are thrust with the pressure of arterial blood. The
result is intravascular hemolysis and the appearance of schistocytes in the peripheral blood film.
Often the creation of thrombi is so brisk that thrombocytopenia results. The disorders associated
with MHA include disseminated intravascular coagulation (DIC), thrombotic thrombocytope-

nic purpura (TTP), hemolytic uremic syndrome (HUS), and the pregnancy-associated syndrome
of hemolysis, elevated liver enzymes, and low platelets (HELLP). A similar clinical picture can
be created by malignant hypertension and macrovascular red cell trauma caused by mechanical
heart valves.
Diagnosis
The peripheral blood smear shows schistocytes and, usually, thrombocytopenia. The associated
conditions are clinicopathologic diagnoses for which there is no single diagnostic test.
Glucose-6-phosphate Dehydrogenase (G6PD) Deficiency

Description
This is the most common red cell enzyme defect. Since red cells lack a nucleus, they lack the capac-
ity to make new enzymes. Even normal red cells have greater enzymatic capacity when young than
when old. However, if the activity of a critical enzyme significantly degrades before the average red
cell life span (120 days), then the cell dies prematurely. Red cells rely on G6PD to produce glutathi-
one that absorbs oxidant stress to protect Hb from oxidation. Oxidized Hb forms precipitates within
the red cell, known as Heinz bodies, whose excision by splenic macrophages results in bite cells.
There are numerous defective forms (disease-causing alleles) of G6PD. Most abnormal alleles
result in a functionally normal enzyme but have a shortened life span within the red cell. Uncom-
mon alleles result in decreased G6PD production, and even young cells have low activity in these
cases. In most forms of the disease, young red cells, especially reticulocytes, have normal G6PD
activity, whereas, in other forms, enzyme activity is universally decreased. Furthermore, the mag-
nitude of this decrease varies. This heterogeneity results in 3 classes of G6PD deficiency: class 1,
in which there is chronic low-level hemolysis; class 2, in which there is profound intravascular
hemolysis following oxidant stress; and class 3, in which there is mild-to-moderate intravascular
hemolysis following oxidant stress.
Most G6PD-deficient persons are clinically well until exposed to excess oxidant (class 2 or 3).
Such exposures arise in the form of ingestion (eg, fava beans), medication use (eg, nitrofurantoin,
antimalarials, sulfa drugs), or infection. In most individuals, there is preferential destruction of
older red cells.
Most G6PD-deficient Diagnosis

persons are clinically The peripheral smear shows a combination of bite cells and Heinz bodies. The latter require spe-
well until exposed to cial (supravital) staining in order to be visualized. Laboratory assays are available for measuring
excess oxidant (class 2 G6PD activity. G6PD activity may appear normal during an acute episode, because only nonhe-
or 3). Such exposures
molyzed, younger cells are available to be assayed. If a normal result is obtained, consider repeat-
arise in the form of
ing the assay in 3 months.
ingestion (eg, fava
beans), medication
use (eg, nitrofurantoin, Pyruvate Kinase (PK) Deficiency
antimalarials, sulfa drugs),
or infection. Description
A steady generation of ATP is needed to maintain the integrity of the red cell membrane. Red cells
generate ATP principally via the glycolytic pathway, in which PK is an active enzyme. Deficient ATP
production leads to progressive red cell dessication, causing predominantly extravascular hemolysis.
PK deficiency is usually a recessively inherited condition. The disease is worldwide in dis-
tribution but slightly more concentrated in particular populations, notably people of Northern
Europe descent and the Pennsylvania Amish.
Diagnosis
Echinocytes are the classic peripheral smear finding, but these appear in large numbers only after
splenectomy. An autohemolysis test is positive and corrects with the addition of ATP. A fluores-
cent spot test is performed in which red cells are incubated with NADH (which fluoresces) to
check for conversion to NAD (which does not).
Paroxysmal Nocturnal Hemoglobinuria

Description
Complement activation occurs at a low level continuously in the blood, and formed C3b that
does not bind to an available surface (bacterium, leukocyte, platelet, or RBC) is rapidly degraded.
Bound C3b can proceed to induce lysis of the cell to which it is attached. Thus, blood cells must
have a mechanism for regularly shedding C3b to avoid lysis.
PNH is due to an acquired (somatic) mutation in the PIG-A gene of a hematopoietic stem
cell, the major consequence of which is decreased production of the glycosylphosphatidylinositol
(GPI) anchor. This is a molecule that functions as a transmembrane anchor for several surface
proteins, many of which are involved in protecting the cell from complement lysis. Affected cells
have decreased expression of, among others, CD16 (the F(c) receptor type III), CD55 (decay- PNH is due to an acquired
accelerating factor [DAF]), and CD59 (membrane inhibitor of reactive lysis [MIRL]). Since this (somatic) mutation
defect is found within an early stem cell, all cell lines (red cells, white cells, platelets) are affected. in the PIG-A gene of a
PNH manifests as hemolysis, and its severity oscillates. Hemoglobinuria reflects the intravas- hematopoietic stem cell,
cular nature of the hemolysis. While hemolysis tends to be episodic (paroxysmal), some patients the major consequence
experience chronic hemolysis of uniform intensity. Furthermore, exacerbations are not usually of which is decreased
nocturnal as implied in the original description. PNH is associated with a thrombotic tendency production of the glyco-
that can be the initial manifestation. Over time the disease may evolve to or present as aplastic sylphosphatidylinositol
anemia. (GPI) anchor.
Diagnosis
A sucrose hemolysis test or acidified serum (Ham) test may be used to screen for PNH. In
these assays, patient blood is exposed to an environment that promotes complement activation.
Enhanced hemolysis in the patient sample as compared with a normal control is interpreted as a
positive test. The preferred diagnostic modality is flow cytometry, a test that allows quantitation
of the surface proteins known to be diminished in PNH.
Sideroblastic Anemia
Description
In the developing erythrocyte, it is within mitochondria that iron is incorporated into porphyrin
to make heme. Ringed sideroblasts are the morphologic expression of the abnormal sequestra-
tion of iron within mitochondria. When increased ringed sideroblasts are found, the differential
diagnosis includes myelodysplastic syndrome, alcohol abuse, copper deficiency (Wilson disease),
lead toxicity, medication effect (isoniazid, pyrazinamide), pyridoxine (vitamin B6) deficiency, and
hereditary sideroblastic anemia.
Diagnosis
In the peripheral blood, one finds anemia with a dimorphic red cell population; that is, there
are normocytic macrocytes and hypochromic microcytes. The diagnosis of sideroblastic anemia
requires a bone marrow biopsy. Nonringed sideroblasts, defined as red cell precursors with 1
to 4 faint siderotic granules, are normal in the marrow. Ringed sideroblasts are defined as red
cell precursors with at least 10 siderotic granules that surround at least one third of the nucleus.
Although they may be seen in small number in some normal individuals and in a wide range of
disorders, when they are found in >15% of all red cell precursors, the diagnosis of sideroblastic
anemia is made.
Pure Red Cell Aplasia

Description
Aplastic anemia is a term that refers to the complete absence of hematopoiesis, affecting granu-
locytic precursors, erythroid precursors, and megakaryocytes. A proliferative disorder may be
isolated to a single cell line, however, as is the case in pure red cell aplasia. This condition may be
acquired or congenital. Acquired pure red cell aplasia may be due to thymoma, EPO therapy, or
infection with parvovirus B19.
Parvovirus B19 may cause a transient arrest of red cell production in healthy children and
adults without serious consequences. Infection usually lasts about 2 weeks, and in those with a
normal red cell life span of 120 days, this usually goes unnoticed. However, in those with chronic
hemolytic anemia, a transient arrest of erythropoiesis may be catastrophic. The virus infects
erythroid progenitor cells, causing a maturation arrest at the pronormoblast stage. Marrow exam-
ination finds numerous giant pronormoblasts, a reduction of the more mature forms, and viral
nuclear inclusions.
Congenital pure red cell aplasia (Blackfan–Diamond syndrome) is a rare, constitutional red
cell aplasia, which usually becomes evident by the age of 5 years. Erythroid precursors in the mar-
row are typically low or absent. HbF is increased.
Diagnosis
The diagnosis is made in a patient with isolated anemia, which is usually normocytic, with reticu-
locytopenia. The bone marrow biopsy shows an isolated decrement in erythropoiesis.
ERYTHROCYTOSIS
Definition
Erythrocytosis was traditionally defined as persistent elevation in the RBC mass. Since RBC mass
is not easily measured, the current WHO definition relies on Hb, specifically Hb above 18.5 g/dL
in men and 16.5 g/dL in women.
Differential Diagnosis
The primary considerations are myeloproliferative neoplasms (MPNs; polycythemia vera [PV]),
reactive (secondary) erythrocytosis, and spurious erythrocytosis due to dehydration (Gaisbock
syndrome) (Table 10–14). Secondary polycythemia is associated with low Pao2 states (such as
smoking and living at high altitudes), abnormal Hb variants, and certain neoplasms (renal cell
carcinoma, cerebellar hemangioblastoma) that produce elevated EPO.
Polycythemia Vera
Definition
PV is an MPN that is due to a clonal neoplastic proliferation of erythroid precursors. It presents
at a mean age of 60, most commonly with hypertension, thrombosis, pruritus, erythromelalgia,
or headache. The erythrocytosis is usually normocytic. Neutrophilia and basophilia are common,
and sometimes thrombocytosis is present. The cause of death is most commonly thrombosis.
Some patients, however, progress to acute leukemia.
TABLE 10–14 Polycythemia Vera Versus Secondary Erythrocytosis

Parameter Polycythemia Vera Secondary Erythrocytosis
RBC mass ↑ ↑
PaO2 Normal Normal to ↓
Leukocytes and basophils Normal to ↑ Normal
LAP score ↑ Normal
Serum vitamin B12 ↑ Normal
Serum EPO ↓ ↑
Serum iron/stainable iron ↓ Normal

LAP, leukocyte alkaline phosphatase; EPO, erythropoietin.
TABLE 10–15 Criteria for Polycythemia Vera: A1 + A2 + Any Other A Criterion Or

A1 + A2 + Any 2 B Criteria
A1 Increased RBC mass or Hb >18.5 g/dL (men), >16.5 g/dL (women)
A2 Erythrocytosis is primary—no familial erythrocytosis, hypoxemia (PaO2 <92%), high-affinity

hemoglobin variant, truncated erythropoietin (EPO) receptor, or tumor that is producing EPO
A3 Splenomegaly
A4 Clonal cytogenetic abnormality other than Philadelphia chromosome
A5 Endogenous erythroid colony formation in vitro
B1 Thrombocytosis >400 × 106/μL
B2 WBC >12 × 106/μL
B3 Panmyelosis on bone marrow biopsy
B4 Low serum EPO
Diagnosis
The diagnosis of PV is made according to strict criteria (Table 10–15). The RBC mass is measured
using isotope dilution, in which a sample of patient red cells is labeled with a radioactive isotope
and reinfused. The red cell mass can then be calculated from the degree of dilution of the labeled
red cells. This direct measurement of the red cell mass distinguishes reduced plasma volume from
a true absolute erythrocytosis.
Examination of the bone marrow shows a marked expansion of erythroid precursors. In PV,
erythroid precursors are capable of spontaneous erythroid colony formation in vitro. In testing
for endogenous erythroid colony formation, patient marrow is cultured. In PV, one can observe
the spontaneous formation of erythroid colonies (without addition of EPO). In healthy patients
or those with secondary erythrocytosis, erythroid colony formation requires exogenous EPO.
The janus kinase 2 (JAK-2) mutation has now been identified in >80% of PV cases. The JAK-2 The janus kinase 2 (JAK-2)
mutation is a valine to phenylalanine substitution at codon 617 (Val617Phe) that appears to con- mutation has now been
fer cytokine-independent growth to cells bearing it. identified in >80% of PV
cases.
METHODS
Red Cell Indices
Measurement of total Hb is carried out most commonly through a chemical reaction. In the
cyanohemoglobin (hemiglobin cyanide [HiCN]) method, Hb is converted to HiCN whose con-
centration is measured by spectrophotometry. The absorbance of the solution at 540 nm reflects
the amount of Hb originally present.
The Hct can be measured directly (manual technique), by centrifuging a tube of whole blood.
The ratio of the packed red cell column height to the total height is the Hct. Note that the Hct is a
unitless value (a percentage), as the units cancel out in its calculation.
Erythrocytes (as well as leukocytes and platelets) can be counted manually, through the use
of a hemocytometer. This is a labor-intensive method that is still used when, for various rea-
sons, the automated analyzer gives erroneous results. RBC counts may be given in terms of cells
per mm3 (eg, 5.5 × 106/mm3), per μL (conventional units), or per L (SI units). When the Hct
and RBC count are determined manually, the remaining red cell indices can be calculated. For
example, MCV = Hct × 1000/RBC. The MCV is stated in femtoliters.
Automated techniques are widely used in clinical laboratories. On most instruments, the red
cell count (RBC), MCV, and RDW are measured directly (as is the total Hb). The instrument then
calculates the other indices, such as Hct. There are at least 3 different methods used by automated
instruments to count cells: impedance (counts any particle of given size), optical methods (light
scatter), or combination of impedance and light scatter.
In impedance counting, cells are suspended in a conductive diluent and passed one-by-
one through an aperture across which a current is flowing. A cell within the aperture causes a
momentary increase in electrical resistance (impedance). Voltage, a product of resistance and

current (V = I × R), increases when the resistance increases. The instrument interprets a momen-
tary increase in voltage as a single cell. The amount of voltage change is proportional to the size
of the cell. The instrument is programmed to count particles measuring between 36 and 360 fL as
red cells. Of course, leukocytes, which are within this size range, will be counted as erythrocytes,
but their relative number is so small (usually) that their effect is typically negligible. RBCs passing
through the aperture come in a range of sizes (volumes), distributed in a roughly Gaussian curve.
The mean of this distribution is taken as the MCV. The variance in the curve is the RDW. The rest
of the red cell indices can be calculated as follows: Hct = MCV × RBC; MCHC = Hb/Hct × 100.
Reticulocyte Counting
Reticulocytes can be measured manually or by automation. In the manual method, a blood smear
is stained with a supravital dye (eg, new methylene blue) that highlights the endoplasmic reticu-
lum that persists within reticulocytes. Red cells and reticulocytes are counted, and the result is
given as a percentage (number of reticulocytes per 100 red cells).
Automated methods are more accurate, since many more cells can be counted. A blood sam-
ple is incubated with a supravital dye, and then passed through an automated counter in which
they are exposed to a laser. Light scatter (which will be greatest in stained reticulocytes) is used to
enumerate the reticulocytes.
Normally reticulocytes constitute less than 1.5% of all red cells. This proportion increases
when red cells are lost or destroyed peripherally, reflecting a marrow response. A normal reticu-
locyte count in the face of anemia is indicative of an impaired marrow.
However, the reticulocyte percentage can be somewhat misleading in the presence of anemia.
This is because anemia leads to increased EPO production, and EPO stimulates the proliferation
of red cell precursors and stimulates the release of reticulocytes from the marrow. Even if the
marrow capacity for proliferation is impaired, the latter effect can produce a transient appearance
of reticulocytosis. Thus, to correct for this, one can calculate the reticulocyte index (RI): RI =
reticulocyte percentage × (patient’s HCT/normal HCT). A normal RI is <3%.
Hemoglobin Electrophoresis
Electrophoresis is the separation of proteins through the application of voltage. Most proteins
have a net charge, usually a net negative charge, and when placed into a semisolid medium (a gel)
will move in response to a voltage. The distance that a protein moves depends on its size and the
magnitude of its charge, so that different proteins can be separated from one another. The posi-
tively charged electrode attracts negatively charged proteins and is called the anode. Proteins that
end up closest to the anode are called fast-migrating or anodal. Proteins that end up farthest from
the anode are considered slow-migrating or cathodal.
If RBCs are lysed, the predominant protein within the lysate is Hb. In the normal adult, this
Hb is largely HbA, with about 2% to 3% HbA2. When this lysate is applied to a gel across which
a voltage is applied, the result is a prominent band (HbA) near the anode (fast-migrating) and a
dim band (HbA2) near the cathode. Any deviation from this pattern is indicative of a hemoglo-
binopathy or thalassemia. Routine Hb electrophoresis is performed by placing a sample of lysed
If RBCs are lysed, the blood on a cellulose acetate gel at pH 8.6 (alkaline electrophoresis). The gel is subjected to elec-
predominant protein tromotive force, fixed, and stained.
within the lysate is Hb. In Thalassemia, being a quantitative defect in production of entirely normal Hbs, does not pro-
the normal adult, this Hb duce abnormal bands on the electrophoresis. Instead, β-thalassemia is diagnosed by the presence
is largely HbA, with about of “thalassemic indices” (low Hct, increased RBC count, low MCV) and a quantitatively increased
2% to 3% HbA2. HbA2. α-Thalassemia has “thalassemic indices” and normal HbA2.
True hemoglobinopathies are due to production of a structurally abnormal Hb molecule that
usually produces a distinct band on electrophoresis. The identity of most, but not all, abnormal
Hbs can be determined by routine electrophoresis, particularly when supplemented with some
clinical information and CBC data. When there is uncertainty, electrophoresis on citrate agar at
pH 6.2 (acid electrophoresis) produces a different set of electrophoretic positions that, in combi-
nation with the alkaline gel, can help identify an abnormal band.
Screening Tests for Sickle Hemoglobin

Rapid detection of sickling Hb, without having to perform electrophoresis, is possible with 1 of
2 types of assay. In the first, the Hb solubility (dithionate) test, one can detect insoluble forms of
Hb within a lysate of blood. Red cells are lysed in sodium dithionate buffer with saponin. After
several minutes, marked turbidity indicates a positive screen. Note that this test detects free Hb Rapid detection of sickling
with altered solubility and may be positive in a number of different genotypes: SS, SA, SC, SD, Hb, without having to
and some types of HbC. This test may be negative when the concentration of HbS is too small, for perform electrophoresis,
example, in neonates. is possible with 1 of
The second type of screening test, the sickling (metabisulfite) test, detects red cells with sick- 2 types of assay.
ling Hbs. In this test, whole blood is subjected to metabisulfite, which encourages cells containing
HbS to sickle. A smear is then examined microscopically for sickling. Like the solubility test, this
test does not give genotypic information and may be positive in SS, SA, SC, S-other, and some
types of HbC. The test requires at least 10% HbS to be positive. Thus, it may not be positive in
neonates or those very aggressively transfused.
Osmotic Fragility Test

All red cells expand and eventually undergo lysis in a hypotonic environment, and spherocytic
red cells do so at a faster rate than normal biconcave red cells. This is the basis of the osmotic fra-
gility test. Red cells are incubated in progressively more hypotonic solutions, parallel with normal
controls. Enhanced lysis, as compared with controls, is a positive test. A positive osmotic fragil-
ity test is not diagnostic of HS, however, since red cells that are spherocytic from any cause will
give a positive result. The most common acquired cause of red cell spherocytosis is autoimmune
hemolytic anemia.
Direct Antiglobulin Test (Coombs Test)

The reagent used in this test is an antibody, obtained from rabbit or goat, that reacts with (binds to)
human globulins (antihuman globulin [AHG]). Specifically, these antibodies may have reactivity
with IgG, complement protein C3, or both. Patient blood is mixed with AHG and then observed
for agglutination (clumping). Depending on the reagent used, agglutination suggests that the
patient’s red cells are coated with IgG, C3, or both. Furthermore, since these were not added to
the red cells in vitro, agglutination indicates that coating occurred in vivo.
In the usual case of WAIHA, the red cells agglutinate mainly with anti-IgG. There may or may
not be reactivity with anti-C3. In cold agglutinin disease, red cells agglutinate only with anti-C3.
While the titer of a warm autoantibody provides little useful clinical information, the titer of a
cold agglutinin can be helpful. The cold agglutinin titer is the highest dilution of patient serum
that causes agglutination of normal RBCs.
Kleihauer–Betke Test
The Kleihauer–Betke (acid elution) test is based on the observation that a weak acid is capable
of eluting normal HbA out of red cells. In contrast, HbF is resistant to acid elution and remains
within red cells. Thus, if blood is subjected to a weak acid, and then smeared and stained, the cells
containing HbA will appear as pale “ghosts,” whereas cells containing HbF appear bright red. In a
pregnant woman, the presence of red cells with HbF is indicative of a fetomaternal hemorrhage.
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C H A P T E R
Bleeding and Thrombotic

Disorders
Elizabeth M. Van Cott and Michael Laposata 11
LEARNING OBJECTIVES
1. Learn the basic molecular events in clot formation and fibrinolysis.
2. Understand the basic classification of disorders in hemostasis.
3. Identify the appropriate laboratory tests for evaluation of the bleeding patient
and the thrombotic patient.
4. Learn the prominent clinical and laboratory features of the individual
disorders of hemostasis.
CHAPTER OUTLINE
Introduction to Hemostasis 254 Drug-induced Immunologic
Clot Formation 254 Thrombocytopenia 272
Fibrinolysis 258 Posttransfusion Purpura 273
Bleeding Disorders 259 Neonatal Alloimmune Thrombocytopenia
Fibrinogen Deficiencies 259 (NAIT) 274
Prothrombin (Factor II) Deficiency 262 Essential Thrombocythemia 275
Factor V Deficiency 262 von Willebrand Disease 275
Factor VII Deficiency 262 Bernard–Soulier Disease and Glanzmann
Hemophilia A (Factor VIII Deficiency) 264 Thrombasthenia 278
Factor VIII Inhibitors 264 Platelet Storage Pool Disease 278
Hemophilia B (Factor IX Deficiency) 265 Hemostatic Defects in Uremia 280
Factor X Deficiency 266 Drug-induced Qualitative Platelet
Factor XI Deficiency 266 Dysfunction 280
Deficiencies of the Contact Factors 267 Thrombotic Disorders 281
Factor XIII Deficiency 267 Hypercoagulable States 281
Antiplasmin Deficiency 267 Antiphospholipid Antibodies: The Lupus
Vitamin K Deficiency 268 Anticoagulant, Anticardiolipin Antibodies,
Disseminated Intravascular Coagulation 269 and Beta-2 Glycoprotein I Antibodies 284
Hemostatic Abnormalities in Thrombotic Thrombocytopenic Purpura 285
Liver Disease 270 Hemolytic–Uremic Syndrome 286
Immune Thrombocytopenic Anticoagulant Therapies 287
Purpura (ITP) 271 Antiplatelet Therapies 288
T
he coagulopathies are grouped into disorders of bleeding and thrombosis. The hemor-
rhagic diseases are further subdivided into the 2 major categories of coagulation factor
disorders and platelet disorders. To understand the diseases with abnormal coagula-
tion that follow, a brief introduction to normal hemostasis precedes the discussions of the
diseases.
253
254 CHAPTER 11 Bleeding and Thrombotic Disorders
INTRODUCTION TO HEMOSTASIS
Normal hemostasis is the controlled activation of coagulation factors and platelets leading to
clot formation, with subsequent clot lysis, in a process that stops hemorrhage without excess
clotting (thrombosis). Effective hemostasis is a rapid and localized response to an interruption
in vascular integrity (vessel wall injury), such that clots are formed only when and where they
are needed.
Clot Formation
Clot formation involves platelet activation and the subsequent generation of fibrin via the coagu-
lation cascade. The 2 processes are discussed separately in the sections that follow.
Platelet Plug Formation

Platelet plug formation is initiated in vivo by exposure of platelets to vascular subendothelium
when a vessel is injured. The platelets adhere to the subendothelium, spread out along the surface,
Clot formation involves and release substances that promote the aggregation of other platelets at that site. The platelets
platelet activation also accelerate fibrin clot formation by providing a reactive surface for several steps in the coagu-
and the subsequent lation cascade.
generation of fibrin via Adhesion of platelets to the subendothelial surface is facilitated by a plasma protein,
the coagulation cascade. von Willebrand factor (vWF), especially in vessels with high shear forces (eg, the fast blood flow
in arteries has a higher shear force than slow blood flow in veins). vWF binds to a specific receptor
on the platelet surface. Deficiency of vWF results in poor adherence of platelets to subendothe-
lium. The severity of bleeding in von Willebrand disease (vWD) varies widely among patients.
Another related platelet adhesion defect occurs in patients whose platelets lack the receptor for
vWF. This bleeding disorder, known as Bernard–Soulier disease, results from an inability of plate-
lets to bind vWF.
Platelet activation occurs from interaction of platelet agonists, most of which are soluble,
with specific receptors on the platelet membrane. Physiologically important agonists include
adenosine diphosphate (ADP), thrombin, epinephrine, collagen, and thromboxane A2, which is
derived from arachidonic acid. A sequence of membrane and cytoplasmic events is initiated by
the agonist–receptor interaction, involving an increase in cytoplasmic calcium ion concentra-
tion and a platelet shape change from a disc to a spiny sphere. The change in cytoplasmic cal-
cium concentration leads to contractile events in the platelet, causing alpha and delta granules
(also known as dense bodies) to fuse with the platelet plasma membrane and release their gran-
ule contents into the extracellular space. Successful granule release requires the formation of
thromboxane A2 from endogenous arachidonate via the enzyme cyclooxygenase. This enzyme
is inhibited irreversibly by aspirin and inhibited reversibly by a number of other anti-inflam-
matory agents. Treatment with aspirin can cause a platelet secretion defect (reduced release of
granule contents) that is often clinically significant in patients with underlying coagulopathies.
Alpha granules contain vWF, fibrinogen, Factor V, 2 platelet-specific proteins—platelet fac-
tor 4 (PF4) and beta-thromboglobulin—as well as a number of other proteins. Delta granules
contain serotonin, adenosine triphosphate (ATP), ADP, pyrophosphate, polyphosphate, and
calcium. The release of some of these substances, in particular ADP, activates unstimulated
platelets nearby. Absence or deficiency of alpha or delta granules occurs as a feature of several
congenital and acquired platelet function disorders, collectively known as storage pool disor-
ders. Individuals whose platelets possess appropriate numbers of intact granules that cannot be
released on appropriate stimulation have a platelet release defect, and on that basis also may
have a bleeding tendency. Aspirin is a common cause of a platelet release defect, because it
impairs thromboxane production.
Release of platelet granule contents is followed by platelet aggregation, that is, the binding of
platelets to one another to form the platelet plug. Normal aggregation requires fibrinogen binding
to platelets via the fibrinogen receptor, which is the glycoprotein IIb/IIIa complex (GP IIb/IIIa),
on the platelet surface. Congenital absence of GP IIb/IIIa results in a bleeding diathesis known as
Glanzmann thrombasthenia (GT).
CHAPTER 11 Bleeding and Thrombotic Disorders 255
α-KAL HK/PK VII/III (Tissue factor)

Xa, IIa
XII XIIa VIIa/III (Tissue factor)
XI/HK IIa XIa
Ca++
IX IXa
Ca++
VIII VIIIa
PL
Intrinsic pathway IIa Extrinsic pathway
X Xa
Ca++ Va V
PL
IIa
II IIa
(Prothrombin) (Thrombin)
I Fibrin
(Fibrinogen)
XIIIa XIII
Cross-linked IIa
Common pathway fibrin
FIGURE 11–1 The coagulation cascade. α-KAL, alpha-Kallikrein; PK, prekallikrein; HK, high-
molecular-weight kininogen; PL, phospholipid.
The platelet surface serves as a site for certain coagulation pathway enzyme reactions (see
below). For example, the platelet membrane can bind the Factor Xa/Factor Va complex that acti-
vates prothrombin to thrombin. Thus, platelet activation and fibrin formation via the coagulation
cascade are interactive biological processes.
Fibrin Clot Formation

The coagulation factor pathway is an enzymatic cascade with sequential conversion of proen-
zymes (zymogens) to fully activated enzymes, which then convert other zymogens to their acti-
vated forms (Figure 11–1). The final steps directly preceding fibrin formation can be activated
through both the intrinsic and extrinsic pathways; hence, this part of the pathway is called the
common pathway. Numerous positive and negative feedback mechanisms exist in the coagulation
pathways so that the cascades do not proceed in an uncontrolled fashion. The pathways are now
known to be extremely complex, with multiple interactions between factors in the intrinsic,
extrinsic, and common pathways. The following description is a version of coagulation factor
interactions that highlights the fundamental reactions.
The coagulation cascade is activated by the appearance of tissue factor (also historically
known as Factor III), which is not normally exposed. Tissue factor is presented when a blood
vessel is injured. It binds with Factor VII and small amounts of circulating active Factor VII
(Factor VIIa), resulting in a complex of Factor VII or VIIa and tissue factor. The Factor VII
in the complex can be autoconverted to Factor VIIa, resulting in a greater number of Factor
VIIa–tissue factor complexes. This complex activates Factor IX to Factor IXa in the intrinsic
pathway, with some activation of Factor X to Factor Xa in the common pathway. Factor IXa,
with support from Factor VIII as a cofactor (or Factor VIIIa, which is a much more effective
cofactor), activates Factor X to Factor Xa. Factor Xa with the assistance of Factor V or, more
effectively Factor Va, converts prothrombin (Factor II) to thrombin (Factor IIa). At this point,
the coagulation cascade is markedly amplified because thrombin activates Factor VIII to Factor
VIIIa, a more effective cofactor, and activates Factor V to its more effective Factor Va form. In
addition, thrombin activates Factor XI to Factor XIa, which, like Factor VIIa and tissue factor,
activates Factor IX to Factor IXa. Thrombin catalyzes the conversion of fibrinogen to fibrin,
which is then cross-linked by Factor XIII to create a stabilized form of fibrin (clot). Factor XIII
is also activated to Factor XIIIa by thrombin.
Factor XII, prekallikrein (PK), and high-molecular-weight kininogen (HMWK, shown as
HK in Figure 11–1) are not required for the generation of fibrin in vivo because even when they
are completely absent, there is no increased risk for bleeding. Nevertheless, the intrinsic path-
way is activated when Factor XII contacts collagen (exposed by vessel injury) or polyphosphate
(released from activated platelets).
This cascade, as it is currently understood and described above, explains 2 long-standing
clinical observations. First, it explains the significant bleeding tendency associated with Factors
VIII and IX deficiencies because these factors are important in the early stages of cascade ampli-
fication. Factor VIIa and tissue factor activate Factor IX to Factor IXa, and Factor IXa requires
Factor VIII to convert Factor X to Factor Xa. Second, this scheme provides an explanation for the
clinical observation that deficiencies of Factor XII, PK, and HMWK are not associated with an
increased risk for bleeding because Factor VIIa/TF activates Factor IX, and thrombin activates
Factor XI, bypassing the need for Factor XII.
Two of the coagulation cascade reactions occur on the platelet surface. The first of these
is the activation of Factor X to Factor Xa, which is produced by platelet-bound Factor IXa and
platelet-bound Factor VIIIa. In the second, platelet-bound Factor Xa and platelet-bound Factor
Va convert prothrombin (Factor II) to thrombin (Factor IIa) in a subsequent step in the coagula-
There are 2 major tion sequence. As noted below, single factor deficiency states, most of which are congenital, exist
inhibitory pathways for all of the factors, but multiple factor deficiencies, which are usually not congenital, are much
that determine the rate more commonly encountered. Inhibitors, as antibodies directed against a specific coagulation
at which the cascade factor, can arise to any of the individual factors to create deficiency states. With some exceptions,
is amplified. One of most factor inhibitors are rare.
these is the protein There are 2 major inhibitory pathways that determine the rate at which the cascade is ampli-
C–protein S anticoagulant fied. One of these is the protein C–protein S anticoagulant pathway (Figure 11–2). As shown in
pathway. An additional the figure, excess thrombin, which is generated through the activation of the coagulation cascade,
mechanism for control of
provides the signal to shut off the coagulation cascade by binding to a protein on the endothelial
the coagulation cascade
surface known as thrombomodulin. The thrombin/thrombomodulin complex converts protein C
involves the inhibitory
into its activated form. The activated protein C then binds free protein S as a cofactor. Protein S
action of antithrombin.
may be bound to C4b-binding protein and to a limited number of other proteins, in which case
protein S becomes inactive. Once free (unbound) protein S binds to activated protein C, the acti-
vated protein C/protein S complex then proteolytically degrades Factors Va and VIIIa, reducing
the flux through the coagulation cascade by removing these 2 activated cofactors. A mutation
in the Factor V molecule, known as the Factor V Leiden mutation, makes Factor V resistant to
proteolytic degradation by the activated protein C/protein S complex. This condition is known as
activated protein C resistance. This permits the prothrombotic action of Factor Va to persist and
contribute to a hypercoagulable state.
An additional mechanism for control of the coagulation cascade involves the inhibitory
action of antithrombin (formerly known as antithrombin III) (Figure 11–3). Antithrombin has a
limited anticoagulant effect on its own, but in the presence of heparin or selected other negatively
charged heparin-like molecules, antithrombin adopts a new conformation that increases its
inhibitory activity 1000-fold, permitting it to inhibit most of the activated coagulation factors
in complexes where both antithrombin and heparin bind to the activated coagulation factor.
Inhibition of Factor Xa, however, does not require the direct binding of the activated coagula-
tion factor by heparin. Factor Xa can be neutralized when it is bound only to antithrombin, after
antithrombin has been activated by heparin or related molecule. The antithrombotic action of
short chains of heparin (low-molecular-weight heparin) is directed primarily against Factor Xa
Va VIIIa
Va & VIIIa degraded

by proteolysis
Activated protein C/
protein S complex
Free protein S
C4b (active) Activated protein C
binding
protein
Bound
protein S Protein C
(inactive)
Thrombin
Activated protein C
Thrombin Thrombin
TM TM TM
Endothelium
FIGURE 11–2 The protein C–protein S anticoagulant pathway. TM, thrombomodulin. (Redrawn
with permission from Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable states.
Hematol Oncol Clin North Am. 1998;12:1141–1166.)
Antithrombin
+ AT + AT conformational
+ + change
– –
– –
Heparin Heparin
Thrombin
(IIa) Inhibition of
Inhibition of + AT
+ AT Xa + thrombin (factor IIa)
– + factor Xa –
– –
–
Low molecular weight heparin Heparin
FIGURE 11–3 The anticoagulant action of antithrombin. AT, antithrombin. (Modified with
permission from Kabakibi A, et al. The hypercoagulable state. Turnaround Times [a newsletter for
physicians at the Massachusetts General Hospital, Boston]. 1994;3:1:1.)
because short heparin chains inhibit predominantly only Factor Xa (longer chains are required
for Factor IIa inhibition). Fondaparinux, a synthetic heparin-related pentasaccharide, inhibits
Factor Xa exclusively.
It should be noted that all of the coagulation factors in the coagulation cascade are syn-
thesized in the liver, and that the activity of Factors II, VII, IX, and X is vitamin K-dependent.
Tissue factor is constitutively expressed on some cell types, and in other cell types, such as
endothelial cells, it is not normally expressed. There is little (if any) tissue factor in the circulat-
ing plasma under normal conditions. It is not exclusively synthesized in the liver. Tissue fac-
tor in the subendothelium is exposed to blood when blood vessels are injured, triggering the
Fibrinolysis is the extrinsic pathway of the coagulation cascade. The half-lives of the coagulation factors in the
controlled dissolution blood vary markedly, with Factor VII showing the shortest half-life of approximately 5 hours
of the formed clot. It and Factor XIII having the longest half-life of more than 120 hours. There is also a wide range
occurs when the injured of plasma concentrations for the coagulation factors. Factor VII is in the lowest concentration
vessel begins to heal, of the circulating coagulation factors at 100 to 500 ng/mL. The highest concentration is found
and is initiated to a for fibrinogen at 200 to 400 mg/dL.
limited extent when clot
formation begins. In this
way, fibrinolysis serves as
a regulatory mechanism Fibrinolysis
to limit excess clot Fibrinolysis is the controlled dissolution of the formed clot. It occurs when the injured vessel
formation. begins to heal, and is initiated to a limited extent when clot formation begins. In this way, fibrino-
lysis serves as a regulatory mechanism to limit excess clot formation.
The principal enzyme involved in fibrinolysis is plasmin, which exists in a zymogen form
known as plasminogen (Figure 11–4). Plasminogen is converted into plasmin by tissue plas-
minogen activator (tPA). Plasmin degrades the fibrin clot. A recombinant form of tPA is used
as a pharmacologic agent to produce thrombolysis (breakdown of a thrombus), in patients with
myocardial infarction or stroke, and clots elsewhere in the body. Clinically effective derivatives
of tPA from genetic manipulation are also used for thrombolysis. tPA is released by endothe-
lial cells, and its secretion into the plasma is increased by thrombin. Plasminogen activator
inhibitors are secreted by platelets and endothelial cells, particularly when they are activated.
Plasminogen activator inhibitors stabilize a newly formed clot by blocking the action of tPA,
thereby preventing premature dissolution of the clot. Plasmin degrades fibrin polymers, and,
to a limited degree, fibrinogen as well, by specific and sequential proteolytic cleavages, generat-
ing fibrin degradation products (FDP). These FDP (fragments X, Y, D, D-dimer, and E) may
be detected in the plasma of patients experiencing fibrinolysis. Deficiency of plasminogen may
predispose to thrombosis, and deficiency of plasminogen activator inhibitor may increase the
risk for bleeding. Antiplasmin inhibits plasmin, but only when it is circulating and not when
Tissue plasminogen activator
+
–
Plasminogen Plasmin Antiplasmin
Plasminogen activator inhibitors
Fibrin
Fibrin degradation
products
FIGURE 11–4 The fibrinolytic pathway. A plus sign indicates that tissue plasminogen activator
converts plasminogen into plasmin. A minus sign indicates inhibitory action. (Redrawn with
permission from Kabakibi, A et al. The hypercoagulable state. Turnaround Times [a newsletter for
physicians at the Massachusetts General Hospital, Boston]. 1994;3:1:1.)
it is clot-bound. This prevents the circulation of a proteolytically active form of plasmin, while
permitting clot lysis to proceed by plasmin within the clot. Deficiency of antiplasmin may result
in a hemorrhagic tendency.
BLEEDING DISORDERS
Figure 11–5 provides a classification of coagulation disorders. The major division in the classifi-
cation is between disorders associated with bleeding and disorders associated with thrombosis.
There are 2 major subdivisions of bleeding disorders—those associated with coagulation factor
and fibrinolytic pathway factor deficiencies, and those associated with an abnormal platelet
count or impaired platelet function. Isolated factor deficiencies are usually congenital, although
occasionally an isolated acquired factor deficiency develops. An example of an acquired iso-
lated coagulation factor deficiency is the Factor X deficiency associated with amyloidosis. The
deficiency of antiplasmin is listed in this section because its absence permits increased plasmin
activity and overactive clot dissolution, resulting in a bleeding tendency. Another major cat-
egory of coagulation factor abnormalities is multiple coagulation factor deficiencies. There are
several commonly encountered situations associated with multiple factor deficiencies. These
include vitamin K deficiency or warfarin intake (which results in a reduced amount of func-
tional Factors II, VII, IX, and X as well as protein C and protein S); disseminated intravascular Quantitative platelet
coagulation (DIC), which results in the consumption of multiple coagulation factors; and liver disorders include
disease that results in decreased synthesis of coagulation factors. Several activated coagulation thrombocytopenia
factors are inhibited by heparin. Heparin administration results in inactivation of most of the and thrombocytosis.
activated coagulation factors. Qualitative platelet
The group of disorders associated with platelets is divided first into quantitative platelet dis- disorders are characterized
orders and qualitative platelet disorders. Quantitative platelet disorders include thrombocytopenia by abnormal platelet
and thrombocytosis. Thrombocytopenia can be produced as a result of increased platelet destruc- function in the presence
of a normal platelet count.
tion, from a variety of immune or nonimmune causes, or decreased platelet production. Com-
mon causes of decreased platelet production include tumor infiltration of bone marrow from
metastases or a hematologic malignancy, and drug-induced thrombocytopenia as occurs with
chemotherapy. Thrombocytopenia can also occur as a result of increased sequestration of plate-
lets in the spleen, usually in patients with splenomegaly. Thrombocytosis is much less common
than thrombocytopenia. Thrombocytosis can be divided into reactive thrombocytosis, in which
there is a transiently increased number of platelets from a stimulus to increase platelet produc-
tion, or neoplastic thrombocytosis, as seen in myeloproliferative disease and, less commonly,
myelodysplastic disorders.
Qualitative platelet disorders are characterized by abnormal platelet function in the presence
of a normal platelet count. vWD is a disorder in which there is defective platelet function, but
from a defect originating outside the platelet, since vWF is generated primarily in endothelial
cells. vWF coats the surface of the activated platelet to allow it to adhere to the cut vessel surface
and initiate platelet plug formation.
Other causes of defective platelet function result from abnormalities within the platelet. These
disorders may be congenital or acquired. The congenital ones are extremely rare, and the acquired
ones are very frequently encountered. Congenital platelet abnormalities associated with defective
function include GT, Bernard–Soulier disease, and storage pool disease (SPD). The much more
common acquired qualitative platelet disorders include drug-induced platelet dysfunction, such
as produced by aspirin and clopidogrel (Plavix), and uremia-induced platelet dysfunction, which
occurs in patients with impaired renal function.
Fibrinogen Deficiencies
Description
Fibrinogen is produced in the liver by hepatocytes. Abnormalities of fibrinogen production
may be congenital or acquired and, in general, involve either decreased production of a normal
molecule (afibrinogenemia and hypofibrinogenemia) or production of an abnormal molecule
(dysfibrinogenemia) (see Table 11–1).
Coagulation disorders
Bleeding disorders associated with altered platelet

Bleeding disorders associated with coagulation
number (quantitative platelet disorders) or impaired
factor and fibrinolytic pathway factor deficiencies
platelet function (qualitative platelet disorders)
Isolated deficiencies of functional coagulation factors Quantitative platelet disorders: Thrombocytopenias

in the coagulation cascade—congenital and acquired
Thrombocytopenia due to increased platelet
• Fibrinogen (Factor I) deficiency destruction—congenital and acquired
• Prothrombin (Factor II) deficiency
• Factor V deficiency Immune
• Factor VII deficiency • Immune thrombocytopenic purpura (ITP)
• Hemophilia A (Factor VIII deficiency) • Drug-induced immune thrombocytopenia
• Factor VIII inhibitors (Factor VIII deficiency from • Posttransfusion purpura (PTP)
antibody-mediated inhibition of Factor VIII activity) • Neonatal alloimmune thrombocytopenia (NAIT)
• Hemophilia B (Factor IX deficiency)
• Factor X deficiency Nonimmune
• Factor XI deficiency • Disseminated intravascular coagulation (DIC)
• Factor XIII deficiency • Thrombotic thrombocytopenic purpura (TTP)
• Deficiencies of Factor XII, prekallikrein, and high- • Hemolytic–uremic syndrome (HUS)
molecular-weight kininogen (contact factors) are • Hypersplenism
not associated with bleeding
Thrombocytopenia due to decreased platelet production
Isolated factor deficiencies in the fibrinolytic pathway • Tumor infiltration of bone marrow
• Plasmin inhibitor deficiency • Drug-induced (nonimmune) thrombocytopenia by
chemotherapeutic agents and other agents
• Aplastic anemia
Disorders associated with deficiencies of

multiple functional coagulation factors
• Vitamin K deficiency
• Disseminated intravascular coagulation (DIC)
• Liver disease from multiple etiologies Quantitative platelet disorders: Thrombocytosis
• Overdose of warfarin or heparin
• Essential thrombocythemia or other
myeloproliferative disorder
Disorders associated with thrombosis
Qualitative platelet disorders

Relatively higher incidence Relatively lower incidence Congenital and acquired
• Activated protein C • Protein C or S deficiency • Defective platelet function
resistance (the Factor V • Antithrombin deficiency resulting from a plasma
Leiden and related • Plasminogen deficiency factor deficiency—von
mutations) • Selected dysfibrinogenemias Willebrand disease
• Prothrombin G20210A • Essential thrombocythemia • Bernard–Soulier disease
mutation • Thrombotic thrombo- • Glanzmann thrombasthenia
• Antiphospholipid cytopenic purpura (TTP) • Storage pool disease
antibody syndrome • Hemolytic–uremic • Uremia-induced platelet
• Heparin-induced syndrome (HUS) dysfunction
thrombocytopenia • Markedly elevated • Drug-induced platelet
homocysteine dysfunction (as produced by
aspirin and other agents)
FIGURE 11–5 A classification of coagulation disorders.

TABLE 11–1 Laboratory Evaluation for Fibrinogen Deficiencies

Results/Comments
Quantitative Deficiencies Dysfibrinogenemia (Qualitative Deficiencies)
Laboratory Test Afibrinogenemia Hypofibrinogenemia Homozygous Heterozygous
PT Markedly prolonged Normal to slightly Markedly prolonged Slightly prolonged

prolonged to normal
PTT Markedly prolonged Normal to slightly Markedly prolonged Slightly prolonged

prolonged to normal
Functional fibrinogen Low Slightly low to normal Low Slightly low to normal
Immunologic fibrinogen Low Slightly low to normal Normal Normal
Thrombin time Prolonged Normal to prolonged Prolonged Prolonged
Reptilase time Prolonged Normal to prolonged Prolonged Prolonged

PT, prothrombin time; PTT, partial thromboplastin time.
In congenital afibrinogenemia and hypofibrinogenemia, there is a reduced (hypofibrinogen-

emia) or absent (afibrinogenemia) production of a normal fibrinogen molecule. In general, the
homozygous deficiency results in afibrinogenemia, and the heterozygous state results in hypofibri-
nogenemia. Both disorders are rare. Homozygotes suffer a mild-to-moderate spontaneous bleed-
ing tendency. Manifestations include umbilical stump hemorrhage and bleeding from mucous
membranes, among many other possible signs and symptoms related to blood loss. Severe bleed-
ing may occur with trauma or surgery. Hypofibrinogenemic patients are usually asymptomatic,
but may bleed significantly with surgery or trauma.
Congenital dysfibrinogenemia is a result of inheritance of a gene for an abnormal fibrinogen
molecule, which is produced in normal or near-normal quantities. All the fibrinogen produced
by a homozygote for dysfibrinogenemia is abnormal, and approximately half of the fibrinogen Acquired
in a heterozygote is abnormal. Hundreds of abnormal fibrinogens have been described. The true hypofibrinogenemia is
incidence of dysfibrinogenemia is not known because many forms of the disorder are asymptom- observed predominantly
atic. Homozygotes may have a mild bleeding tendency, perhaps because the fibrinogen molecule in patients with advanced
is cleaved too slowly to form fibrin monomers or because abnormal fibrin monomers polymerize liver disease, in patients
too slowly. The bleeding tendency is characterized by easy or spontaneous bruising, menorrhagia, with a consumptive
and prolonged or severe bleeding with surgery or trauma. Heterozygotes are usually asymptom- coagulation disorder
atic, but may show excessive bleeding with surgery or trauma. Several types of dysfibrinogenemia such as DIC, and in those
(approximately 10%-15% of cases) are associated with an increased risk of thrombosis rather treated with thrombolytic
therapy.
than bleeding. A few types of congenital dysfibrinogenemia are associated with both bleeding
and thrombosis.
Acquired hypofibrinogenemia is observed predominantly in patients with advanced liver dis-
ease, in patients with a consumptive coagulation disorder such as DIC, and in those treated with
thrombolytic therapy.
Acquired dysfibrinogenemia represents the acquired production of an abnormal fibrinogen
molecule in normal or near-normal quantities, most often in patients with acute or chronic liver
disease, especially those with primary or metastatic hepatic malignancies. The patient may or may
not be symptomatic, depending on 1) whether there is simultaneous production of normal fibrin-
ogen in amounts sufficient to allow normal hemostasis and 2) whether the abnormal fibrinogen
can polymerize like a normal fibrinogen molecule (see the section “Hemostatic Abnormalities in
Liver Disease”).
Diagnosis
See Table 11–1 for the laboratory evaluation of the patient with a fibrinogen deficiency.
Prothrombin (Factor II) Deficiency

Description
Prothrombin (Factor II) is the precursor to thrombin (Factor IIa), which converts fibrinogen
into fibrin in the common pathway of the coagulation cascade. Deficiency of prothrombin,
either inherited or acquired, may result in a hemorrhagic diathesis. Inherited abnormalities of
prothrombin are rare. As with fibrinogen, abnormalities occur in 2 major forms. The first is
reduced or absent production of a normal prothrombin molecule. The second is production of
normal amounts of an abnormal prothrombin molecule with decreased activity (dysfunctional
form or dysprothrombinemia). Heterozygotes usually have approximately 50% of normal activ-
ity and may be asymptomatic or have a bleeding tendency. In 1 study, 83% of a small cohort
of heterozygotes had bleeding, with Factor II levels ranging from 21% to 35%. Homozygotes
Abnormalities occur usually have 1% to 25% of normal activity and have a mild-to-severe hemorrhagic tendency.
in 2 major forms. The
Acquired hypoprothrombinemia occurs most often along with deficiencies of Factors VII, IX,
first is reduced or
and X in vitamin K deficiency and with warfarin (Coumadin) therapy; with deficiencies of mul-
absent production of
a normal prothrombin
tiple coagulation factors in liver disease or DIC; as an isolated coagulation factor deficiency in
molecule. The second some patients with lupus anticoagulant (LA); and in patients exposed to topical bovine throm-
is production of normal bin who develop antibodies to prothrombin (and not uncommonly to Factor V also). Bleeding
amounts of an abnormal manifestations depend on the level of prothrombin activity; usually no bleeding occurs with a
prothrombin molecule prothrombin level >50% of normal.
with decreased activity
(dysfunctional form or Diagnosis
dysprothrombinemia). See Table 11–2 for the laboratory evaluation of the patient with a prothrombin (Factor II)
deficiency.
Factor V Deficiency
Description
Factor V is a high-molecular-weight protein (approximately 300,000 Da) that acts as an accelerat-
ing cofactor for the enzymatic conversion of prothrombin to thrombin by Factor Xa. When Factor
V is cleaved to Factor Va by thrombin, its cofactor activity is significantly increased. Factors Va
and VIIIa are degraded by activated protein C. An isolated deficiency of Factor V is a rare cause
of bleeding.
Apparent heterozygous and homozygous deficient states have been observed. Heterozy-
gotes usually have levels of approximately 50% of normal and can experience bleeding or may be
asymptomatic. In a cohort of 19 heterozygous patients, 50% had bleeding, with Factor V levels
ranging from 21% to 55%. Homozygotes have variable levels below 50%; they are most likely to
be symptomatic if the level is 10% or less.
As with the other coagulation factors, 2 major forms of the inherited deficiency are described:
reduced or absent production of a normal Factor V molecule (absence form) and production of
an abnormal molecule with reduced activity in normal amounts (dysfunctional form). A rare
combined deficiency of Factors V and VIII is due to a genetic defect in intracellular transport of
Factors V and VIII. Acquired deficiencies of Factor V occur with liver dysfunction or DIC.
Diagnosis
See Table 11–2 for the laboratory evaluation of the patient with a Factor V deficiency.
Factor VII Deficiency

Description
Factor VII is a vitamin K-dependent coagulation factor precursor that, when activated by
thrombin, Factor Xa, or Factor IXa, is converted to Factor VIIa. This activated factor then con-
verts phospholipid-bound Factor X into Factor Xa in the presence of calcium and tissue factor.
It also converts Factor IX to Factor IXa. Factor VII deficiency may occur as an inherited or
acquired disorder.
TABLE 11–2 Laboratory Evaluation for Coagulation Factor Deficiencies

PT Prolonged PTT Prolonged
Deficient Factor(s) If Deficient? If Deficient? Other Tests Useful in Diagnosis/Comments
Fibrinogen—see Table 11–1
Prothrombin Yes Yes (usually less Factor II assay; selected other factor assays to determine if Factor II is low
(Factor II) prominent than the along with other factors, especially VII, IX, and X—this often establishes
PT prolongation) the cause of a low Factor II level
A lupus anticoagulant test to determine if a low Factor II level is associated
with a lupus anticoagulant
Factor V Yes Yes (usually less Factor V assay; selected other factor assays to determine if a low
prominent than the Factor V level is accompanied by other factor deficiencies
PT prolongation)
Factor VII Yes No Factor VII assay; selected other factor assays to determine if Factor VII is
low along with other factors, especially II, V, IX, and X
Factor VIII No Yes Factors VIII and IX assays, as these 2 deficiency states are clinically
indistinguishable
von Willebrand factor antigen and ristocetin cofactor to determine if a low
Factor VIII level represents hemophilia A or von Willebrand disease
Factor IX No Yes Factors VIII and IX assays as these 2 deficiency states are clinically
indistinguishable; selected other factor assays to determine if Factor IX
is low along with other factors, especially II, VII, and X
Factor X Yes Yes (usually less Factor X assay; selected other factor assays to determine if Factor X is low
prominent than the along with other factors, especially II, VII, and IX
PT prolongation)
Factor XI No Yes Factor XI assay
Factor XII No Yes Factor XII assay
Prekallikrein No Yes Prekallikrein assay
High-molecular- No Yes High-molecular-weight kininogen assay

weight kininogen
Factor XIII No No The screening test for Factor XIII deficiency assesses the solubility of
the patient’s clot in urea—clots from patients with Factor XIII levels
<2% dissolve; a quantitative assay not involving clot solubility is also
available and can detect mild deficiencies of Factor XIII
The inherited deficiency state, which is rare, may be present as reduced or absent production
of a normal molecule (absence form) or production of an abnormal molecule with decreased
activity in normal amounts (dysfunctional form). An inherited isolated deficiency of Factor VII The bleeding risk is
in heterozygotes is usually associated with a Factor VII activity level of approximately 50%. In a difficult to predict
cohort of 88 heterozygous patients, 36% had a bleeding tendency, with Factor VII levels ranging because the factor VII
from 21% to 69%. In homozygotes, there are variable Factor VII activity levels below 50%. The activity level correlates
bleeding risk is difficult to predict in these patients because the factor activity level correlates poorly with the patient’s
poorly with the patient’s tendency to hemorrhage, but in general values <10% can be associated tendency to hemorrhage.
with major spontaneous bleeding. A large proportion of patients with less than 2% Factor VII A large proportion of
do not bleed. Acquired Factor VII deficiency occurs in vitamin K deficiency and with warfarin patients with less than
therapy along with deficiencies of Factors II, IX, and X; and in DIC or liver disease along with 2% Factor VII do not
multiple other coagulation factor deficiencies. bleed.
Intracranial hemorrhage has been reported in Factor VII-deficient patients, most often occur-
ring in infants <1 year of age. Elevated Factor VII levels have been associated with an increased
risk of cardiovascular disease.
Diagnosis
See Table 11–2 for the laboratory evaluation of the patient with a Factor VII deficiency.
Hemophilia A (Factor VIII Deficiency)

Description
Hemophilia A is a bleeding disorder resulting from a deficiency of Factor VIII procoagulant
activity. Factor VIII circulates in the plasma bound to vWF. Approximately 90% of patients with
hemophilia A synthesize low amounts of normal Factor VIII molecules, and 10% of patients with
hemophilia A synthesize normal amounts of an abnormal (nonfunctional) Factor VIII. Hemo-
philia A is inherited as an X-linked trait, and 65% to 75% of patients have a positive family history.
Disease prevalence in the United States is 1 in 10,000 males; the carrier state in females is rarely
symptomatic. Hemophilia A and hemophilia B (Factor IX deficiency, see below) are clinically
indistinguishable. The likelihood of hemorrhage depends on the amount of Factor VIII present;
the majority of patients (approximately 50%-70% of hemophilia A patients) have severe disease.
The severity of disease is categorized as follows:
• In mild disease: the VIII level is 6% to 20% of normal, with rare spontaneous bleeding.
• In moderate disease: the VIII level is 1% to 5% of normal, with occasional spontaneous
bleeding.
• In severe disease: the VIII level is <1% of normal, with frequent spontaneous bleeding.
Hemophilia A is a All hemophilia patients (A and B) may experience severe hemorrhage following trauma or
bleeding disorder surgery if there is no prior treatment to elevate the factor level. Bleeding that is characteristic
resulting from a of hemophilia (A and B) includes intra-articular (joint), intracranial, and intramuscular hemor-
deficiency of Factor VIII rhage. The latter can produce a compartment compression syndrome. Easy bruising and pro-
procoagulant activity. longed bleeding after minor cuts and abrasions are also characteristic. The onset of hemorrhage is
Factor VIII inhibitors are typically delayed following injury, and pathologic bleeding may occur hours after injury. Primary
antibodies, usually IgG, hemostasis (dependent on platelet plug formation) is intact, but secondary hemostasis (dependent
that bind to Factor VIII
on the fibrin clot generated by the coagulation cascade) is defective. Up to 15% of hemophilia A
and inhibit its coagulant
patients develop an inhibitor to Factor VIII at some time during the course of their disease (ie, an
activity.
antibody against Factor VIII). The inhibitor develops only in those transfused with Factor VIII-
containing products, and most often in patients with <1% Factor VIII. Factor VIII inhibitors may
also spontaneously occur rarely in nonhemophiliacs (see the section “Factor VIII Inhibitors”).
Diagnosis
See Table 11–2 for information regarding the laboratory evaluation for Factor VIII deficiency.
Factor VIII Inhibitors

Description
Factor VIII inhibitors are antibodies, usually IgG, that bind to Factor VIII and inhibit its coagu-
lant activity.
Factor VIII inhibitors have been found in several clinical situations.
• Inhibitors are diagnosed most commonly in patients with hemophilia A. Inhibitors
occur in 10% to 15% of these patients and make the treatment of hemorrhage much
more difficult. The vast majority of cases of Factor VIII inhibitors in hemophilia A
patients occur in those with severe hemophilia A (<1% Factor VIII activity). Inhibitor
formation is related to transfusion of exogenous Factor VIII, and usually develops
before 100 treatment days if it appears. Two immune response patterns have been
observed in hemophilia A patients. The first is a high response pattern. Inhibitors
rise to a high titer in response to exposure to Factor VIII. The titer may not decline
for months to years, even without further exposure to Factor VIII. Rapid anamnestic
responses are often seen within 3 to 7 days of reexposure in these patients. In the
second pattern, there is a low response. In addition, inhibitors usually remain at a low
titer despite reexposure. They may occasionally disappear and reappear spontaneously.
Little, if any, anamnestic response is likely found in a low responder.
• Spontaneous inhibitors to Factor VIII can occur in the postpartum patient. Usually they
are recognized 2 to 5 months after the birth of the first child and disappear spontaneously
TABLE 11–3 Laboratory Evaluation for Factor VIII Inhibitor

PT Normal
PTT Prolonged; normalizes in a 1:1 PTT mixing study of patient plasma and normal
plasma with a 0-min incubation (ie, PTT performed immediately after mixing),
but becomes prolonged with a longer incubation of the mixed plasma (60 or
120 min) at 37°C; the PTT of the mixed plasma at 60-120 min incubation with a
clinically significant inhibitor is typically at least 8 s longer than the PTT of the
mixed plasma at 0-min incubation
Factor VIII activity Decreased
Factor VIII inhibitor assay Used for quantitation of inhibitor; inhibitor levels are expressed in Bethesda
units (BU); 1 BU/mL is the amount of inhibitor that produces a 50% reduction
in Factor VIII activity
after 12 to 18 months. However, the course is variable, and there are reports of death
from hemorrhage in some patients. Antigenic differences between mother and fetus do
not sufficiently explain the development of a Factor VIII inhibitor, and its cause remains
unknown.
• Inhibitors may occur in those with allergic and enhanced immunologic reactions,
including patients with:
(a) Rheumatoid arthritis
(b) Systemic lupus erythematosus
(c) Reactions to drugs, such as penicillin, chloramphenicol, sulfonamides, and phenytoin
(d) Malignancy
(e) Asthma
(f) Crohn disease
(g) Ulcerative colitis
(h) Pemphigus
(i) Multiple myeloma
• Inhibitors may appear in patients without any obvious underlying disorder. These are
usually older individuals, and the inhibitor may remit in several months, persist for years,
or disappear with immunosuppressive therapy.
In a hemophilia A patient, a poor response to treatment with Factor VIII concentrate may
be the first indication that an inhibitor is present, or there may be an increased frequency of
bleeding episodes. In nonhemophiliacs, development of a new hemorrhagic tendency is usually
the presenting feature of a spontaneous Factor VIII inhibitor. The most favorable prognoses are
for patients with low titer inhibitors, peripartum women, and patients without an underlying Hemophilia B is an
disorder. inherited hemorrhagic
disorder resulting from
Diagnosis a lack of procoagulant
See Table 11–3 for information regarding the laboratory evaluation for a Factor VIII inhibitor. activity of Factor IX.
Hemophilia B (Factor IX Deficiency)

Description
Hemophilia B is an inherited hemorrhagic disorder resulting from a lack of procoagulant activity
of Factor IX. Factor IX is a vitamin K-dependent factor that, in its active form (Factor IXa), is a
serine protease of the intrinsic pathway of the coagulation cascade. Approximately 70% to 90% of
hemophilia B patients have a deficiency of a normal coagulant protein, and 10% to 30% produce
an abnormal Factor IX that is nonfunctional. Inheritance is sex-linked, with affected males, and
female carriers. Of hemophilia B patients, 60% to 70% have a positive family history for bleed-
ing. The prevalence of hemophilia B is much less than that of hemophilia A. Approximately 1 in
50,000 males in the United States has hemophilia B versus 1 in 10,000 males with hemophilia A.
The hemophilia B carrier state in the female is usually asymptomatic, as is the case with hemo-
philia A. Acquired Factor IX deficiency may occur along with deficiencies of Factors II, VII, and
X in patients with vitamin K deficiency or those receiving warfarin therapy, and with deficiencies
of other coagulation factors in patients with liver disease, DIC, or nephrotic syndrome.
As previously noted, hemophilia B is clinically indistinguishable from hemophilia A. The
severity of hemorrhage depends on the amount of Factor IX activity present:
• In mild disease: 6% to 20% of normal IX activity is present, with rare spontaneous bleeding.
• In moderate disease: 1% to 5% of normal IX activity is present, with occasional
spontaneous bleeding.
• In severe disease: <1% of normal activity is present, with frequent spontaneous bleeding.
Profuse bleeding may occur in any hemophilia B patient with trauma or surgery if there is
no prior treatment to elevate the factor level. Bleeding in hemophilia B resembles that found in
hemophilia A and includes deep tissue bleeding, intra-articular bleeding (hemarthrosis), intra-
cranial bleeding (which may be lethal), and intramuscular bleeding with potential compartment
compression syndrome. Severe mucosal membrane bleeding can occur in hemophilia, particu-
larly after dental surgery.
Inhibitors develop to Factor IX in 1% to 5% of hemophilia B cases. These antibodies often
occur in high titer and frequently present a major bleeding problem despite treatment.
Diagnosis
See Table 11–2 for information regarding the laboratory evaluation for patients with
hemophilia B.
Factor X Deficiency
Description
An inherited isolated deficiency of Factor X is a rare disorder. Homozygotes and heterozygotes
have been identified. Homozygotes usually possess <2% of normal activity. Heterozygotes usually
possess 40% to 70% of normal activity. In a cohort of 15 heterozygous patients, 33% had a bleed-
ing tendency, with Factor X levels ranging from 23% to 47%. Patients with Factor X values <10%
can have a high risk of spontaneous major bleeding, and those with >40% are usually asymptom-
atic. Inherited Factor X deficiency, like the other factor deficiency states, occurs in 2 major forms:
reduced or absent synthesis of a normal molecule (absence form) and synthesis of an abnormal
molecule in normal amounts (dysfunctional form).
Acquired Factor X deficiency may result from warfarin or vitamin K deficiency (in the pres-
ence of deficiencies of Factors II, VII, and IX), from liver disease (with deficiencies of other fac-
tors synthesized in the liver), with DIC, or as an isolated deficiency in cases of amyloidosis. In
amyloidosis, Factor X becomes irreversibly bound to amyloid fibrils in the extracellular space,
and is thereby removed from the circulation.
Diagnosis
See Table 11–2 for information regarding the laboratory evaluation of patients with Factor X
deficiency.
Factor XI Deficiency
Description
Factor XI deficiency is a commonly encountered disorder. Homozygotes typically have less than
20% of normal Factor XI activity. Heterozygotes have 20% to 70% of normal Factor XI activity.
The deficiency in almost all cases appears to be a reduced or absent production of a normal mol-
ecule, rather than production of an abnormal or dysfunctional molecule.
The vast majority of the cases of Factor XI deficiency are in people of Jewish descent, par-
ticularly those of Ashkenazi origin. The frequency of the homozygous deficient state is 0.2% to
0.3% in the Ashkenazi population, and the frequency of the heterozygous state is extremely high,
at approximately 5.5% to 11.0%.
The hemorrhagic tendency is variable for both heterozygotes and homozygotes. Patients with
Factor XI levels <15% to 20% uncommonly have spontaneous bleeding but frequently have post-
operative bleeding, and patients with levels between 20% and 65% tend to be asymptomatic or
have low rates of postoperative bleeding. Bleeding does not correlate well with the level of Factor
XI activity. Some homozygotes have an abnormal partial thromboplastin time (PTT), a very low
Factor XI level of less than 10%, and no bleeding, even with surgery. The bleeding tendency of a
particular individual is more closely related to the bleeding tendency of the patient’s kindred than
to the measured Factor XI level. The explanation, which is true for all mutations affecting coagula-
tion factors, is that certain mutations produce a low level of Factor XI and a prolonged PTT but
are not clinically significant in vivo. This is because they only affect the activity of the factor in
the in vitro clotting factor assays, which are not exact replicas of clot formation in vivo. Acquired
decreases in Factor XI can occur with pregnancy, proteinuria, liver dysfunction, and DIC.
Diagnosis
See Table 11–2 for information regarding the laboratory evaluation of patients with Factor XI
deficiency.
Deficiencies of the Contact Factors

Description
The contact coagulation factors (so named because they were originally thought to activate the A deficiency of any of the
coagulation cascade by contacting the cut surface of the vessel wall) include Factor XII, PK, and contact factors prolongs
HMWK. A deficiency of any of the contact factors prolongs the PTT because the PTT assay is the PTT because the PTT
constructed to involve these factors, even though the coagulation cascade in vivo does not depend assay is constructed to
on these factors. Bleeding diatheses have not been reported in patients with deficiencies at any involve these factors, even
level of Factor XII, PK, or HMWK. Factor XII deficiency is fairly common, with many thousands though the coagulation
affected, especially individuals of Asian descent and children with tonsillitis. HMWK deficiency cascade in vivo does not
and PK deficiency are rare. depend on these factors.
Bleeding diatheses have
not been reported in
Diagnosis patients with deficiencies
See Table 11–2 for information regarding the laboratory evaluation for contact factor abnormalities. at any level of Factor XII,
PK, or HMWK.
Factor XIII Deficiency
Description
Factor XIII circulates in plasma as a zymogen and is converted to its active form (Factor XIIIa)
by thrombin. Factor XIIIa catalyzes the formation of covalent bonds between chains of adjacent
fibrin monomers. This stabilizes the fibrin clot, making it rigid and more resistant to the action
of plasmin. Congenital deficiency of Factor XIII is rare. The bleeding tendency in homozygotes is
characterized by umbilical stump bleeding in newborns (>90% of patients with clinically signifi-
cant Factor XIII deficiency have this finding), intracranial hemorrhage, miscarriages, and post-
traumatic hematomas, with bleeding often delayed hours to days after the trauma. Patients with
mild or moderate deficiencies might have mucocutaneous bleeding or be asymptomatic. Patients
with Factor XIII levels above 30% are usually always asymptomatic.
Diagnosis
See Table 11–2 for information regarding the laboratory evaluation for Factor XIII deficiency.
Antiplasmin Deficiency
Description
Antiplasmin or plasmin inhibitor (formerly known as alpha-2 antiplasmin) is a glycoprotein
(GP) that serves as a regulator of fibrinolysis in several ways (see Figure 11–4). It blocks the
TABLE 11–4 Laboratory Evaluation for Antiplasmin Deficiency

PT Normal
PTT Normal
Antiplasmin Decreased
enzymatic activity of plasmin (the major fibrinolytic enzyme) and other serine proteases, some
of which are coagulation factors, and it inhibits the binding of plasminogen to fibrin. A bleeding
diathesis is associated with the congenital deficiency of plasmin inhibitor. It is an extremely rare
disorder and only homozygotes with <10% of normal plasmin inhibitor activity appear to be
clinically affected. Those who do bleed may experience mucosal membrane bleeding (particu-
larly in the genitourinary tract), subcutaneous hematomas, spontaneous bruising, and severe
bleeding with trauma. Most heterozygotes are asymptomatic, but those few who are symptom-
atic have only a mild bleeding tendency. Acquired deficiency of plasmin inhibitor can occur in
liver disease, nephrotic syndrome, amyloidosis, DIC, and, most notably, following thrombo-
lytic therapy. In thrombolytic therapy, plasminogen is purposefully converted to plasmin, which
results in the formation of plasmin–antiplasmin complexes, thereby reducing the amount of
available antiplasmin.
Diagnosis
See Table 11–4 for information on the laboratory evaluation of plasmin inhibitor deficiency.
Vitamin K Deficiency
Description
In adults, vitamin K deficiency most often occurs secondary to disease or drug therapy; it rarely
occurs as a dietary deficiency. Causes of vitamin K deficiency include:
In adults, vitamin K • Warfarin therapy (reduces the amount of active vitamin K)

deficiency most often • Antibiotic therapy (capable of suppressing bowel flora that synthesize vitamin K)
occurs secondary to • Malabsorption syndromes: cystic fibrosis, sprue, ulcerative colitis, Crohn disease, parasitic
disease or drug therapy; infections, short bowel syndrome, and ileojejunostomy (for morbid obesity)
it rarely occurs as a • Dietary restriction with incidental decrease in vitamin K intake
dietary deficiency. • Long-term total parenteral nutrition
• Biliary obstruction
Vitamin K depletion can occur in as little as 2 weeks if both intake (enteral and parenteral)
and endogenous production of vitamin K are eliminated. In early deficiency, Factor VII only, or
Factors VII and IX only, may be decreased due to their shorter half-lives. Vitamin K deficiency
may present as an asymptomatic prolongation of the PT in mild cases or as a major spontane-
ous hemorrhage in severe deficiencies. The degree of prolongation of the PT does not accurately
predict the risk of hemorrhage.
Most antibiotics destroy bacterial flora and must be considered as a possible cause of vitamin
K deficiency in the bleeding patient. However, certain cephalosporins produce vitamin K defi-
ciency much more rapidly than other antibiotics. Cephalosporins with an N-methylthiotetrazole
(MTT) group in position 3 directly inhibit the vitamin K-dependent carboxylase that is respon-
sible for converting Factors II, VII, IX, and X to their active form. Cephalosporins in the MTT
group include cefamandole, cefoperazone, cefotetan, moxalactam, cefmetazole, and cefmenox-
ime. Weekly prophylaxis with vitamin K has been recommended when MTT-cephalosporins are
given to patients at high risk for vitamin K deficiency.
TABLE 11–5 Laboratory Evaluation for Vitamin K Deficiency

PT Always prolonged
PTT May be prolonged, depending on severity
Factors II, VII, IX, and X, protein C, The combined deficiencies of Factors II, VII, IX, and X,
and protein S protein C, and protein S are diagnostic of vitamin K
deficiency or ingestion of a vitamin K antagonist, such
as warfarin, especially if a non-vitamin K-dependent
coagulation factor (eg, Factor V) level is normal; Factor VII
and protein C decrease before the others because they
have the shortest half-lives in the plasma
Diagnosis
See Table 11–5 for information on the laboratory evaluation for vitamin K deficiency.
Disseminated Intravascular Coagulation

Description
DIC is a common acquired coagulation disorder that occurs secondary to a variety of underlying
disorders. The most common cause is infection; 10% to 20% of patients with gram-negative sepsis
develop DIC. Other causes of DIC include obstetrical complications (retained dead fetus, placen-
tal abruption, amniotic fluid embolism, hypertonic saline-induced abortion, and septic abortion),
extensive tissue injury (including trauma, ischemia, infarction, and burns), liver disease, transfu-
sion of ABO-incompatible blood, and adult respiratory distress syndrome. The clinical presenta-
tion varies from an asymptomatic condition, detectable only by laboratory abnormalities, to a
severe coagulopathy with a mortality of up to 80%.
The major events in acute DIC, independent of the cause, are microvascular thrombosis The major events in acute
with consumption of platelets and coagulation factors, and then hemorrhage as a result of low DIC, independent of the
levels of platelets and coagulation factors and overactivation of the fibrinolytic system to remove cause, are microvascular
the thrombi. Hemorrhagic symptoms can include any of the following—petechiae, ecchymoses, thrombosis with
mucosal oozing, hematuria, gastrointestinal tract bleeding, bleeding into surgical wounds, and consumption of platelets
prolonged bleeding at venous access sites. Severe bleeding may contribute to hypotensive shock. and coagulation factors,
DIC may present as a more chronic, low-grade condition in patients with malignancy. These and then hemorrhage as
patients are at risk for macrovascular (large vessel) thrombosis as well, most likely as a deep a result of low levels of
vein thrombosis. platelets and coagulation
The prolongations of the PT and the PTT reflect a decrease in fibrinogen and other coagu- factors and overactivation
lation factors that are consumed by clotting. In addition, fibrinogen is degraded by excess of the fibrinolytic system
plasmin activation in the fibrinolytic system. Platelets are also consumed, and, therefore, the to remove the thrombi.
platelet count is typically low. The presence of FDP, 1 of which is the D-dimer, indicates that
fibrin clots have been formed and subsequently degraded. There is no single laboratory test
that can diagnose or exclude DIC, and the diagnosis is made when the characteristic labora-
tory abnormalities are present along with a known stimulus for DIC. A practical approach to
diagnosis of DIC is to perform the PT, the PTT, and a D-dimer assay, with serial measurements
of fibrinogen and platelets. In severe acute DIC, most of the laboratory test results will be
abnormal, although fibrinogen may be normal or even elevated. In chronic DIC, the labora-
tory abnormalities may be less pronounced or even absent because the liver and bone marrow
can increase production of coagulation factors and platelets, respectively, to offset the losses
from consumption.
Diagnosis
See Table 11–6 for information on the laboratory evaluation for DIC.
TABLE 11–6 Commonly Used Assays in the Laboratory Evaluation for

Disseminated Intravascular Coagulation (DIC)
FDP or D-dimer Elevated in essentially all cases, both acute and chronic; elevated due to
fibrinolysis of fibrin deposits in the microvasculature
PT Prolonged in most cases of clinically significant DIC, due to a decrease

in fibrinogen, prothrombin (Factor II), and multiple other coagulation factors
PTT Prolonged less often than the PT in DIC, but increases along with severity
of DIC due to a decrease in fibrinogen, Factor VIII, and multiple other
coagulation factors
Platelet count Decreased in most cases of acute DIC due to consumption of platelets
Fibrinogen Low or decreasing with serial samples in <50% of acute DIC cases, due to
the conversion of fibrinogen into fibrin; the fibrinogen level can be normal
or even elevated in DIC by a variety of mechanisms, 1 of which is increased
fibrinogen synthesis in the liver as part of the acute-phase response to
infection or other stimulus
Schistocytes Present in the peripheral blood smear in approximately 50% of acute DIC
cases; results from microangiopathic hemolysis of RBCs as they traverse
through vessels that are partially occluded by fibrin strands
FDP, fibrin degradation products; PT, prothrombin time; PTT, partial thromboplastin time.
Hemostatic Abnormalities in Liver Disease

Description
Patients with acute and chronic liver disease often have laboratory evidence of a hemostatic
abnormality. These patients may be asymptomatic or have only mild bleeding problems, but those
with advanced liver disease may experience a severe hemorrhage.
Hemorrhage in patients with liver disease may be due to 1 or more of the following:
Patients with acute and • Coagulation factor abnormalities: These are caused by decreased hepatic synthesis of
chronic liver disease vitamin K-dependent factors (II, VII, IX, and X) and non-vitamin K-dependent factors.
often have laboratory Decreased fibrinogen is usually found only in patients with severe hepatic failure; in fact,
evidence of a hemostatic patients with acute hepatitis without hepatic failure usually have an increased fibrinogen
abnormality. These level.
patients may be • Thrombocytopenia: This frequently occurs as a consequence of sequestration in the spleen,
asymptomatic or have impaired platelet production, or increased platelet destruction. It is not usually a severe
only mild bleeding decrease in platelet number.
problems, but those with • Platelet dysfunction: The dysfunction is usually mild and its clinical significance is
advanced liver disease uncertain; platelet dysfunction may be clinically important only in liver disease patients
may experience a severe with severe thrombocytopenia or severe renal failure, which can result in uremia-induced
hemorrhage. platelet dysfunction.
• DIC or a DIC-like syndrome: There is no general agreement as to whether the coagulation
abnormalities that occur in patients with liver disease are due to DIC, liver disease alone,
or a combination of these and other mechanisms. A DIC-like syndrome occurs frequently
in patients with acute hepatic failure. Laboratory abnormalities in these cases include
hypofibrinogenemia, thrombocytopenia, increased FDP such as D-dimer, and decreased
levels of Factors V and VIII.
• Acquired dysfibrinogenemia (in patients with selected liver diseases [see the section
“Fibrinogen Deficiencies”]): Impaired fibrin polymerization may result and thereby
predispose the patient to bleeding.
• Increased fibrinolysis: Hemostatic abnormalities in patients with cirrhosis may be
due to increased fibrinolysis. This may occur as a result of decreased hepatic clearance
of plasminogen activators and decreased synthesis of inhibitors of fibrinolysis
(see Figure 11–4).
TABLE 11–7 Laboratory Evaluation for Hemostatic Defects in Liver Disease

PT and PTT Both will be prolonged, but the PT prolongation is usually greater than the PTT
prolongation
D-dimer or FDP Elevated due to decreased clearance by the liver
Fibrinogen Most often slightly low or normal; can be elevated if underlying illness causes
acute-phase reaction
Platelet count Most often slightly low or normal
Coagulation factor assays Used to investigate the extent of coagulation factor deficiencies; in the absence
of a concomitant DIC
DIC, disseminated intravascular coagulation; FDP, fibrin degradation products; PT, prothrombin time; PTT, partial thromboplastin time.
Diagnosis
The laboratory evaluation for hemostatic defects from liver disease is shown in Table 11–7.
Immune Thrombocytopenic Purpura (ITP)

Description
ITP (where the I formerly stood for idiopathic) exists in both an acute and a chronic form. The
disorder is one in which accelerated platelet destruction occurs in the absence of other causes
such as DIC, thrombotic thrombocytopenic purpura (TTP), drug-induced thrombocytopenia,
and neonatal thrombocytopenia. Platelet production is also often reduced.
The destruction of platelets in ITP is antibody-mediated. The amount of platelet-associated
IgG is increased in the majority of patients with acute and chronic ITP. Many patients with chronic
ITP have increased levels of antiplatelet antibodies in the serum, as well as on the platelet surface.
It should be noted that there are a host of disorders unrelated to immune thrombocytopenias,
which are associated with increased IgG on the platelet surface. Helicobacter pylori infection has
been associated with ITP.
In acute ITP, the platelet may be an innocent target of an antipathogen antibody that
cross-reacts with an epitope on the platelet membrane. Chronic ITP appears to be more of a
classic autoimmune illness in which the target antigens for platelet autoantibodies are platelet
GPs. Sequestration and destruction of antibody-coated platelets occur predominantly in the
spleen.
Acute ITP usually presents as a childhood illness with peak incidence between 2 and 9 years. Acute ITP usually presents
It is heralded by a prodromal illness, such as a viral respiratory infection, in 60% to 80% of cases. as a childhood illness with
The infection occurs 2 to 21 days prior to onset of thrombocytopenia. The risk of hemorrhage is peak incidence between
greatest during the first 1 to 2 weeks after the onset of acute ITP. Intracranial hemorrhage is the 2 and 9 years. Chronic ITP
most feared complication of ITP. The majority of patients experience a spontaneous resolution of occurs most commonly
acute ITP 3 weeks to 3 months after onset. A small percentage of patients do not recover fully after between the ages of
12 months, and advance to a diagnosis of chronic ITP. 20 and 50 years, and in
Chronic ITP occurs most commonly between the ages of 20 and 50 years, and in females females more often than
more often than in males (ratio of 2:1 to 3:1). It is characterized by the absence of a prodromal males.
illness and the presence of mild bleeding that may continue for months before medical attention
is sought. Manifestations include scattered petechiae or purpura, mostly on distal extremities,
mild mucosal bleeding, easy bruising, epistaxis, and menorrhagia. ITP is often discovered in an
asymptomatic patient found to have a low platelet count as part of a complete blood count (CBC).
The diagnosis of ITP is made only after ruling out other causes for an isolated thrombocytopenia
by history, physical examination, and laboratory studies.
Diagnosis
See Table 11–8 for information on the laboratory evaluation for ITP.
TABLE 11–8 Laboratory Evaluation for Immune Thrombocytopenic Purpura (ITP)

Platelet count In acute ITP, most cases have <20,000 platelets/μL; in chronic ITP, counts
range from 5,000 to 75,000/μL commonly, and, on average, are higher than
platelet counts in patients with acute ITP
Platelet morphology Large (young) platelets are often seen on peripheral smear
PT and PTT Normal
Hemoglobin and hematocrit May be low if an accompanying blood loss is severe or long-standing; if
there is no evidence of blood loss but there is an anemia, the possibility
of a concomitant autoimmune hemolytic anemia and thrombocytopenia
(Evans syndrome) should be considered
Antiplatelet antibodies A test for antiplatelet antibodies is not recommended for diagnosis of
ITP; it is generally neither sensitive nor specific for ITP; most patients with
acute or chronic ITP will have increased immunoglobulin on the platelet
surface; however, many disorders have increased levels of platelet-
associated antibodies, including sepsis, drug-induced thrombocytopenia,
lymphoproliferative disorders, disseminated intravascular coagulation, and
autoimmune diseases; the test for antiplatelet antibodies, using various
methodologies, detects small quantities of antibody on the platelet surface
(much less antibody than is found on RBCs in patients with a positive direct
antiglobulin test result)
Bone marrow aspirate Not required unless indicated on the basis of other signs/symptoms.
The bone marrow shows normal RBC and WBC precursors, and a
normal or increased number of megakaryocytes; bone marrow platelet
production may be increased greatly in an attempt to compensate for
rapid platelet destruction
H. pylori, HIV, and HCV testing These infections can cause an ITP-like thrombocytopenia that resolves when
the infection is treated
PT, prothrombin time; PTT, partial thromboplastin time; H. pylori, Helicobacter pylori; HIV, human immunodeficiency virus;
HCV, hepatitis C virus.
Drug-induced Immunologic Thrombocytopenia

Description
Many drugs have been implicated in drug-induced immune thrombocytopenia. However, most
cases can be attributed to relatively few drugs, notably heparin, quinidine/quinine, gold salts,
Many drugs have
and sulfonamides. Exposure to most of these compounds is readily ascertained. However, when
been implicated in obtaining the patient’s history, one should include inquiries regarding consumption of over-the-
drug-induced immune counter medications and topical medications, as well as soft drinks, mixers, and aperitifs to rule
thrombocytopenia. out exposure to quinine. The pathogenesis of thrombocytopenia for most drugs involves both the
However, most cases can drug and IgG (as the predominant class of antibody involved). A plasma protein bound to the
be attributed to relatively drug serves as the antigen; the antigen combines with a specific antibody, and this complex binds
few drugs, notably to the platelet membrane. This is known as an “innocent bystander” effect. The antibody-coated
heparin, quinidine/ platelet is then sequestered and destroyed. Certain other drugs (eg, protamine, bleomycin, and
quinine, gold salts, and ristocetin) can cause destruction of platelets by a direct toxic effect that is nonimmune. In hep-
sulfonamides. arin-induced thrombocytopenia (HIT), a complex of heparin and a circulating protein derived
from the platelet, known as platelet factor 4 (PF4), acts as the antigen. The complex, along with
bound antibody, binds to the platelet surface, causing platelet activation, and unlike other drug-
induced thrombocytopenias, an increased risk for thrombosis.
The true incidence of drug-induced immunologic thrombocytopenia is not known. The inci-
dence varies with the drug in question and the clinical condition or treatment of the patient.
It may be as high as 1% to 3% of people exposed to the drug, as is the case with unfractionated
(standard) heparin. Of quinidine users, approximately 1 in 1000 develop symptomatic thrombo-
cytopenia. Drug-induced immunologic thrombocytopenia occurs most commonly in patients
more than 50 years old, but it also has been reported in infants less than 1 year old. It is not pos-
sible to predict that patients will develop thrombocytopenia from drug treatment.
TABLE 11–9 Laboratory Evaluation for Drug-induced Immunologic

Thrombocytopenia
Platelet count Extremely low (<10,000/μL) to slightly low
Tests for drug-dependent antibodies Not routinely available (except for HIT antibody testing). Both test
bound to the platelet and for drug- results are usually positive; however, they may be negative if the
dependent antiplatelet antibodies in reaction is dependent on an in vivo drug metabolite, rather than
the serum unbound to platelets the parent drug if only the parent drug is used in the laboratory
test; an assay for platelet activation by the drug should be positive
if the mechanism of thrombocytopenia, in the presence of the drug,
includes activation of platelets by antigen–antibody complexes; an
in vivo challenge with the suspected drug to confirm toxicity can
be extremely dangerous and is rarely, if ever, indicated; there are a
variety of methodologies for tests assessing drug-induced platelet
activation, including flow cytometry and the measurement of
serotonin released from activated platelets; an ELISA assay for HIT
detects antibodies to heparin–platelet factor 4 complexes
HIT, heparin-induced thrombocytopenia.
Ingestion of a drug that induces thrombocytopenia may produce flushing, fever, headache,
and chills prior to onset of thrombocytopenia. The onset of thrombocytopenia may be abrupt fol-
lowing drug exposure or, if it requires antibody generation to lower the platelet count, it may be
delayed for 4 to 15 days. Anamnestic responses may occur and if they arise, the delay is shorter.
Bleeding may occur as early as 6 to 12 hours after exposure to the drug in highly responsive
patients. Bleeding manifestations may include 1 or more of the following: petechiae, purpura
(usually the first symptom), mucosal hemorrhagic bullae, gastrointestinal or genitourinary hem-
orrhage, intrapulmonary hemorrhage, and, lastly, intracranial hemorrhage, which is rare, but Heparin-induced
often lethal. HIT is unique in that bleeding is uncommon, and, as noted above, HIT patients are thrombocytopenia
at risk for thrombosis rather than bleeding. is unique in that
bleeding is uncommon,
and HIT patients are at
Diagnosis risk for thrombosis rather
See Table 11–9 for information on the laboratory evaluation for drug-induced immunologic than bleeding.
thrombocytopenia. Laboratory tests for drug-induced thrombocytopenia are not routinely avail-
able, with the exception of testing for HIT. If HIT is considered, a platelet count should be per-
formed first. If the platelet count decreases to 50% or less of its apparent baseline value, a test for
antibodies to the heparin–PF4 complex or a functional test that shows platelet activation in the
presence of heparin and the patient’s plasma should be performed.
Posttransfusion Purpura
Description
Posttransfusion purpura (PTP) is a rare syndrome characterized by the sudden onset of throm-
bocytopenia 7 to 10 days following transfusion of blood or blood products containing platelets or
platelet material. The thrombocytopenia appears to be due to antibody-mediated destruction of
autologous as well as transfused platelets. In over 90% of cases, the antibody that develops in the
affected individuals is directed against the antigen HPA-1a, formerly known as P1A1, on platelet
membrane GP IIIa. In these cases, the recipient’s own platelets are HPA-1a negative. It is not known
why there is destruction of the patient’s own HPA-1a-negative platelets following platelet transfu-
sion with HPA-1a-positive platelets. Only 2% to 3% of the population in the United States lacks this
antigen. Antibodies against other platelet-specific antigens have been reported in PTP, but they are
much less commonly encountered. In almost all cases, the development of anti-HPA-1a antibody is
thought to be an anamnestic response, with prior sensitization occurring through previous trans-
fusion or pregnancy.
PTP occurs predominantly in females, perhaps due to the likelihood of sensitization through
pregnancy. The interval between the first exposure to the HPA-1a antigen and the transfusion
TABLE 11–10 Laboratory Evaluation for Posttransfusion Purpura

PT and PTT Normal
Platelet count Usually decreased to <10,000/μL
Tests for antiplatelet antibodies Positive (see below)

in the serum
Test for HPA-1a antigen on platelets in The HPA-1a antigen is absent from the patient’s platelets;
cases involving the HPA-1a antigen anti-HPA-1a antibody is present in the patient’s serum in the
majority of cases and an attempt should be made to
demonstrate the specificity of this antibody to HPA-1a
that incites the thrombocytopenia is greater than 3 years in most cases in which the information
has been reported. The onset of thrombocytopenia is fulminant in most cases, with the platelet
count decreasing to <10,000/μL. Hemorrhage usually begins with purpura and mucocutaneous
bleeding, and may progress to gastrointestinal and genitourinary bleeding, epistaxis, oozing from
intravenous access sites, and intracranial hemorrhage. In severely affected patients sustained with
supportive therapy of red blood cells and/or platelets, the thrombocytopenia usually begins to
resolve in 14 days (mean value with a range 1-35 days). Cases with less severe thrombocytopenia
apparently require a longer recovery period (24 days average, range 6-70 days). The outcome is
fatal in approximately 10% of cases, usually due to hemorrhage. The risk of fatal hemorrhage
appears to be greatest at the onset of the disease. Recurrent PTP has been documented, with the
recurrence appearing no sooner than 3 years after the first episode, even though antibody may
persist in the patient’s blood during the intervening time and there may be repeated challenges
with exogenous platelets.
Diagnosis
See Table 11–10 for information on the laboratory evaluation for PTP.
Neonatal alloimmune Neonatal Alloimmune Thrombocytopenia (NAIT)

thrombocytopenic
purpura is a disorder in Description
which there is destruction NAIT is a disorder in which there is destruction of platelets in the fetus and newborn. The
of platelets in the fetus destruction occurs following transplacental passage of maternal IgG antibodies directed against
and newborn. The a platelet-specific antigen present on fetal platelets and absent from the mother’s platelets. The
destruction occurs antibody-coated platelets are removed from the circulation by the neonate’s reticuloendothelial
following transplacental system around the time of birth. The estimated incidence ranges from 1 in 5000 to 1 in 2000
passage of maternal births, with an increasingly higher incidence in recent years attributed to improved surveillance
IgG antibodies directed and serologic testing for the disorder. The platelet-specific antigen implicated in 80% to 90% of
against a platelet-specific all cases (and 95% of symptomatic cases) of NAIT is HPA-1a. As previously noted, this antigen is
antigen present on fetal present on the platelets of 97% to 98% of the general population. In approximately 50% of cases,
platelets and absent from NAIT occurs during the first pregnancy; when it does occur, there is a 97% chance that the next
the mother’s platelets.
pregnancy will be affected.
Affected newborns are usually the product of an otherwise uncomplicated pregnancy and
delivery. Within hours after birth, petechiae and ecchymoses appear in a generalized distribu-
tion. Other clinical signs include neurologic abnormalities if intracranial hemorrhage occurs,
and pallor from anemia, if the bleeding is severe. Intracranial hemorrhage is the leading cause of
death in NAIT, with a 50% mortality. Overall mortality from NAIT is approximately 5% to 10%.
Thrombocytopenia usually persists for approximately 2 weeks in untreated cases (range of 1 week
to 2 months), and 1 week in treated cases.
Diagnosis
See Table 11–11 for information on the laboratory evaluation for NAIT.
TABLE 11–11 Laboratory Evaluation for Neonatal Alloimmune

Thrombocytopenia (NAIT)
PT and PTT Normal
Platelet count (in infants) Normal to slightly decreased at birth, continues to decrease after birth
with gradual decline beginning several hours after birth; many cases
show only mild thrombocytopenia, but most symptomatic cases have
<30,000 platelets/μL; approximately 50% have <20,000 platelets/μL;
returns to normal within 2-3 weeks
Hemoglobin/hematocrit May be decreased with hemorrhage
Anti-HPA-1a antibodies A mother with HPA-1a-negative platelets is positive for the anti-HPA-1
(in HPA-1a-related cases) antibody in serum
Discussions of TTP and hemolytic–uremic syndrome (HUS), which are thrombocytopenias

associated with thrombosis, are presented among the thrombotic disorders.
Essential Thrombocythemia
Description
Essential thrombocythemia is a chronic myeloproliferative disorder, characterized by thrombo-
cytosis arising from the clonal proliferation of a neoplastic multipotent stem cell. Life expectancy
can be essentially normal with a median survival of 10 to 15 years, but the disease course is fre-
quently complicated by both hemorrhage and thrombosis. A small percentage (<5%) of patients
progress to acute leukemia, predominantly those patients previously treated with radioactive
phosphorus or alkylating agents to reduce their platelet counts. At the time of diagnosis using
older criteria, mild splenomegaly occurs in 30% to 50% of patients, and hepatomegaly in 15% to
20%. Using 2008 WHO diagnostic criteria, splenomegaly is present in only a minority of patients
at diagnosis. The incidence of the disorder is higher in older age groups.
The principal diagnostic feature of essential thrombocythemia is a persistently elevated plate-
let count with bone marrow megakaryocyte hyperplasia. Patients with this disorder can progress
to a “spent” phase, characterized by myelofibrosis and a low platelet count. The purpose of the
laboratory testing is to eliminate other possible etiologies for the thrombocytosis. Other entities
in the differential diagnosis of an elevated platelet count include reactive thrombocytosis and
other myeloproliferative disorders—myelofibrosis, polycythemia vera, and chronic myelogenous
leukemia.
Diagnosis
See Table 11–12 for information on the laboratory evaluation for essential thrombocythemia.
von Willebrand Disease von Willebrand disease is

caused by a quantitative
Description deficiency of normal von
vWD is caused by a quantitative deficiency of normal vWF in the majority of cases and a quali- Willebrand factor in the
tatively abnormal vWF in the remainder of cases. vWF normally polymerizes to form multimers, majority of cases and a
which are aggregates of a single vWF polypeptide; in normal plasma, the multimers have a range qualitatively abnormal
of sizes. vWF has 2 major roles: von Willebrand factor in
the remainder of cases.
• Platelet adhesion: Large multimers of vWF (ie, those with many units of the single
polypeptide) effectively promote platelet adhesion to the subendothelium in injured
vessels; if only small multimers are present, platelet plugs form poorly.
• Binding of Factor VIII: vWF circulates in the plasma with Factor VIII, the coagulant protein
that is lacking in hemophilia A. vWF prolongs the half-life of Factor VIII by protecting it from
rapid degradation. If vWF is reduced, Factor VIII coagulant activity is often reduced as well.
TABLE 11–12 Laboratory Evaluation for Essential Thrombocythemia (ET)

Platelet count a
Sustained platelet count >450,000/μL
a
Hemoglobin Lack of elevation in hemoglobin helps exclude polycythemia vera
a
DNA testing JAK2 V617F or other clonal marker
a
Absence of BCR-ABL (to exclude chronic myelogenous leukemia)
a
Bone marrow biopsy Proliferation mainly of the megakaryocytic lineage with increased
numbers of enlarged, mature megakaryocytes. No significant increase
or left shift of neutrophil or red cell lineages. Absence of fibrosis helps
exclude myelofibrosis
Platelet aggregation Does not contribute to diagnosis, but platelets may be hypoaggregable or
hyperaggregable
Acute-phase reactants Elevated acute-phase reactants, such as C-reactive protein or fibrinogen,

raise the possibility that the thrombocytosis is reactive rather than ET.
Causes of reactive thrombocytosis include iron deficiency, splenectomy,
surgery, infection, inflammation, connective tissue disease, malignancy,
and other causes (aif JAK2 V617F is absent, the diagnosis requires absence
of evidence for reactive thrombocytosis; however, the presence of reactive
thrombocytosis does not exclude ET if all of the other requirements
are present)
a
Required for diagnosis according to 2008 WHO guidelines.
vWD prevalence estimates vWD prevalence estimates vary, with reported values as high as 1% of the general population.
vary, with reported Unlike hemophilia A and B, vWD affects both men and women. It is likely to be the most com-
values as high as 1% of mon inherited bleeding disorder. There are 3 major types of vWD. The types were reorganized
the general population. and renumbered with Arabic numerals in the 1990s (see Table 11–13). The most common type
Unlike hemophilia A and (type 1) is usually a mild bleeding disorder; it accounts for the majority of all cases of vWD. Type
B, vWD affects both men 2 vWD includes patients with qualitative vWF defects. Type 3 is rare and inherited as an autoso-
and women. mal recessive trait. It is associated with severe bleeding and very low to absent vWF levels. The
types are distinguished from each other by laboratory testing.
TABLE 11–13 Classification of von Willebrand Disease (vWD)

Type and Description of Defect
1: Partial quantitative deficiency of vWF
2: Qualitative defects in vWF
2A: Absence of high-molecular-weight multimers in plasma due to a defect in vWF
2B: Absence of high-molecular-weight multimers due to increased affinity of abnormal vWF for platelet
glycoprotein Ib
2M: Decreased vWF function without the loss of high-molecular-weight multimers
2N: Decreased affinity for Factor VIII (can be misdiagnosed as hemophilia)
3: Severe quantitative deficiency of vWF
Platelet-type vWD: Absence of high-molecular-weight multimers in plasma due to increased affinity of

abnormal platelet vWF receptor for normal vWF
Acquired vWD: Reduction in plasma vWF associated with the presence of an underlying disease that leads to
removal of vWF from the circulation
vWF, von Willebrand factor.
TABLE 11–14 Laboratory Evaluation for von Willebrand Disease (vWD)

Ristocetin cofactor activity A functional assay for vWF; assesses the ability of normal platelets
(vWF:RCo) to aggregate in the presence of ristocetin; normal aggregability to
ristocetin requires large multimers of vWF
von Willebrand factor antigen An immunologic assay for vWF; assesses the quantity (not the
(vWF:Ag) function) of vWF
Factor VIII activity (Factor VIII) Factor VIII becomes decreased secondary to the low vWF; if it is
low enough, decreased Factor VIII activity is associated with a
prolonged PTT
vWF multimer analysis vWF multimer analysis assesses the size distribution of vWF multimers;
loss of high-molecular-weight multimers occurs in type 2A and type
2B vWD
Blood Group Mean vWF (%)

O 75
A 106
B 117
AB 123
PTT, partial thromboplastin time; vWF, von Willebrand factor.
It should also be noted that the mean vWF levels vary with blood type as shown in the second
portion of Table 11–14.
More than 65% of patients with vWD have type O, presumably because patients with this
blood type start from a lower baseline value for vWF.
The severity of bleeding is highly variable among patients, even within the same subtype of
vWD, and even within an individual patient over time. Typically, bleeding manifestations such
as easy bruising or epistaxis begin in early childhood. Other manifestations include menorrhagia
and mucous membrane bleeding (from the gingiva, oropharynx, and gastrointestinal and geni-
tourinary tracts). Profuse hemorrhage may occur with a significant hemostatic challenge such as
trauma or surgery.
Diagnosis
Laboratory test results vary with the type and subtype of vWD. Like the severity of bleeding, the If the patient history
laboratory values can also vary widely over time for an individual patient, and may sometimes be strongly suggests vWD,
normal. Normal results from a single determination do not rule out the diagnosis. If the patient and the test results are
history strongly suggests vWD, and the test results are normal, the tests should be repeated at a normal, the tests should
later time because plasma levels for vWF are increased during pregnancy, stress, while receiving be repeated at a later time
oral contraceptives, and during an acute illness or injury. Therefore, values obtained at these times because plasma levels
may be unreliable for diagnosis. It is also not yet clear if the absolute level of vWF or the level for vWF are increased
relative to the mean vWF for the blood type of the patient is more important in establishing the during pregnancy, stress,
while receiving oral
diagnosis. Current guidelines note that the absolute level of vWF seems to be more important
contraceptives, and during
than the level relative to blood type.
an acute illness or injury.
See Table 11–14 for information on the laboratory evaluation for vWD.
von Willebrand Disease Types, Subtypes, and

Their Expected Test Results
• Type 1: vWF multimers of all sizes are decreased due to a defect in synthesis or release
of vWF from the endothelium, the site of most vWF synthesis. Functional (ristocetin
cofactor or vWF:RCo) and antigenic (vWF antigen or vWF:Ag) levels of vWF are usually
proportionately decreased. Factor VIII activity might also be low. The vWF multimer
pattern shows a normal distribution of multimers.
• Type 2A: Absence of large and intermediate-size vWF multimers from the plasma and
platelet surface, due to a defect in the synthesis or polymerization of multimers, or from
increased proteolysis of multimers. Functional activity (vWF:RCo) is decreased compared
with antigenic levels (vWF:Ag). Therefore, vWF:RCo < vWF:Ag < Factor VIII is the most
commonly observed pattern in type 2A. The vWF multimer pattern shows an abnormal
distribution of multimers, with the absence of large and intermediate-size multimers.
• Type 2B: Marked deficiency of large vWF multimers from plasma. Intermediate-size and
small multimers are present. Large multimers are present on the patient’s platelets, due to
increased affinity of the abnormal vWF molecule for the platelet surface. Functional and
antigenic levels in plasma samples are similar to those in type 2A (vWF:RCo < vWF:Ag <
Factor VIII). The vWF multimer pattern shows the absence of large multimers from plasma.
The patient’s platelets show increased aggregation at low concentrations of ristocetin that do
not cause normal platelets to aggregate. The patient’s platelets aggregate at low concentrations
of ristocetin because they are coated with large vWF multimers.
• Types 2M and 2N are uncommon and are briefly described in Table 11–13.
• Type 3: Severe deficiency of all vWF multimers, due to a marked defect in synthesis. Factor
VIII activity is less severely affected than vWF activity. Both functional and antigenic vWF
levels are markedly reduced. The vWF multimer pattern shows a virtual absence of all-size
multimers.
• Platelet-type Willebrand disease: vWF is qualitatively normal, but abnormal platelets have
an increased affinity for large multimers of vWF due to a defect in platelet GP Ib. The
laboratory test values are similar to those in type 2B.
• Acquired vWD: This disorder has been found in patients with systemic lupus
erythematosus, multiple myeloma, Waldenström macroglobulinemia, lymphoproliferative
disorders, and other diseases. Patients have no congenital or familial history of bleeding.
Causes of the decrease in circulating vWF include adsorption of large multimers onto cells
(eg, lymphocytes or tumor cells) or the presence of antibodies to vWF. Acquired vWD
resolves when the underlying disorder is effectively treated.
Bernard–Soulier Bernard–Soulier Disease and Glanzmann Thrombasthenia

syndrome and Glanzmann
thrombasthenia are rare
Description
congenital hemorrhagic Bernard–Soulier syndrome (BS) and GT are rare congenital hemorrhagic disorders that result
disorders that result from absent or defective specific platelet membrane GPs, impairing platelet function. BS is char-
from absent or defective acterized by a decrease of functional GP Ib/IX/V, the platelet receptor for vWF. GT is character-
specific platelet ized by a decrease of functional GP IIb/IIIa, the complex that mediates platelet aggregation by
membrane glycoproteins, binding fibrinogen to the platelet surface when platelets are activated.
impairing platelet GT often decreases in severity with age. Manifestations include easy bruising, epistaxis,
function. mucous membrane bleeding—particularly in the gastrointestinal tract—and menorrhagia. The
amount of hemorrhage is highly variable among affected patients.
Diagnosis
See Table 11–15 for information on the laboratory evaluation for BS and GT.
Platelet Storage Pool Disease

Description
Platelet SPD represents a group of disorders in which there is a deficiency of platelet granules.
Decreased secretion of platelet granular contents at the time of platelet activation makes the
platelets less hemostatically effective. The congenital forms of SPD include:
• Delta SPD: platelets have a decreased number of delta (dense) granules; these secretory
granules contain ADP, polyphosphate, serotonin, and calcium.
• Alpha-delta or alpha-partial delta SPD: decreased number of delta granules with either
a complete or partial deficiency of alpha granules; alpha granules contain many proteins
including fibrinogen, PF4, platelet-derived growth factor, and beta-thromboglobulin.
TABLE 11–15 Laboratory Evaluation for Bernard–Soulier Disease and Glanzmann

Thrombasthenia
Platelet count Slightly to moderately decreased in BS, occasionally normal; in GT, the
platelet count is usually normal, but may be slightly decreased
Platelet morphology on In BS, the platelets are very large (>80% of platelets are >2.5 mm in
peripheral smear diameter) accounting for the synonym for this disorder as the “giant
platelet syndrome”; in GT, platelets usually appear normal
PT and PTT Normal
Platelet aggregation studies In BS, there is decreased aggregation with ristocetin, but a normal
response with epinephrine, ADP, arachidonic acid, and collagen; in GT,
aggregation is absent (with nearly flat line tracings) with epinephrine,
ADP, collagen, and arachidonic acid, but there is normal aggregation
with ristocetin
Quantitative tests for platelet BS patients have low amounts of glycoprotein Ib/IX; GT patients have
glycoproteins low amounts of glycoprotein IIb/IIIa
ADP, adenosine diphosphate; BS, Bernard–Soulier disease; GT, Glanzmann thrombasthenia; PT, prothrombin time;
PTT, partial thromboplastin time.
• Alpha SPD (“gray platelet syndrome”): decreased number of alpha granules, and a
normal number of delta granules; platelets appear gray, large, and vacuolated on a
peripheral blood smear.
SPD also may occur as an acquired abnormality, acutely in patients who have been supported
on a cardiopulmonary bypass device and chronically in some cases of acute leukemia and myelo-
proliferative disorders. The molecular basis of most types of congenital SPD is unknown. It may
result from abnormal granule morphogenesis or abnormal granule maturation in megakaryo-
cytes. SPD may be a manifestation of a global defect in granule formation as in the Hermansky–
Pudlak syndrome (see below). Hereditary SPD is the most common congenital qualitative platelet Most patients with
disorder, but it is still quite rare. storage pool disease
Most patients with SPD have mild bleeding symptoms. Bleeding manifestations of SPD have mild bleeding
include mild mucous membrane bleeding, easy bruising, menorrhagia, and excessive bleed- symptoms. Bleeding
ing following dental or general surgery. SPD may also occur as a component of the following manifestations of SPD
syndromes: include mild mucous
membrane bleeding, easy
• Hermansky–Pudlak syndrome: Features include delta SPD, oculocutaneous albinism, bruising, menorrhagia,
pulmonary fibrosis, and the accumulation of ceroid-like material in cells of the and excessive bleeding
reticuloendothelial system. One subtype is due to a defective gene (called HSP1) on following dental or
chromosome 10. general surgery.
• Chediak–Higashi syndrome: Features include delta SPD with giant platelet granules,
photophobia, nystagmus, pseudoalbinism, lymphadenopathy, splenomegaly, and
increased susceptibility to infection. It is attributed to defects in a gene called CHS1 on
chromosome 1, affecting protein trafficking.
• Thrombocytopenia with absent radius: Features include alpha SPD and absence of the
radius bone.
• Wiskott–Aldrich syndrome: Features of this X-linked recessive disorder include delta SPD
with other metabolic platelet defects, recurrent infections, eczema, lymphocytopenia,
multiple cellular and humoral immunologic defects, and thrombocytopenia with
microplatelets (small platelets); the thrombocytopenia may resolve following splenectomy.
It is attributed to a genetic defect in a gene called WASP on the X chromosome, affecting
signal transduction and other functions.
Diagnosis
See Table 11–16 for information on the laboratory evaluation for storage pool deficiency.
TABLE 11–16 Laboratory Evaluation for Storage Pool Deficiency

PT and PTT Normal
Platelet count Variable
Peripheral blood smear Shows thrombocytopenia with large, gray, vacuolated platelets in alpha
SPD; giant granules in platelets, neutrophils, eosinophils, lymphocytes,
and monocytes in Chediak–Higashi syndrome; microplatelets and
thrombocytopenia in Wiskott–Aldrich syndrome
Platelet aggregation Absent or extreme diminution of the secondary wave of aggregation with
studies ADP and epinephrine
Platelet ATP/ADP ratio Increase in delta granule deficiency due to low ADP in these platelets (this
testing is not routinely available)
Electron microscopy of May reveal a decrease in alpha granules, delta granules, or both
circulating platelets
Alpha granule quantitation Alpha granule contents may be assayed by measuring the amount of platelet
beta-thromboglobulin or platelet factor 4, both of which are normally present
in alpha granules (this testing is not routinely available)
ADP, adenosine diphosphate; ATP, adenosine triphosphate; PT, prothrombin time; PTT, partial thromboplastin time; SPD,
storage pool deficiency.
The bleeding tendency Hemostatic Defects in Uremia

in uremia-induced
hemorrhage is attributed Description
to platelet dysfunction The bleeding tendency in uremia-induced hemorrhage is attributed to platelet dysfunction and
and endothelial cell endothelial cell dysfunction.
dysfunction. Bleeding Bleeding manifestations may be mild or severe and can include petechiae, ecchymoses,
manifestations may epistaxis, and purpura. Paradoxically, chronic renal failure is also associated with an increased
be mild or severe and incidence of arterial and venous thrombosis, and, therefore, can influence hemostasis toward
can include petechiae, bleeding or clotting.
ecchymoses, epistaxis,
and purpura. Diagnosis
See Table 11–17 for information on the laboratory evaluation for hemostatic defects in uremia.
Drug-induced Qualitative Platelet Dysfunction

Description
Platelet dysfunction may occur on ingestion of a wide variety of drugs, particularly aspirin and
clopidogrel (Plavix). Due to the ubiquity of aspirin in over-the-counter medications, many medi-
cations are implicated in platelet dysfunction. Some patients consume multiple drugs, such as
aspirin and clopidogrel, with different and additive antiplatelet effects and thereby inhibit plate-
let function by more than 1 mechanism. Drug-induced platelet dysfunction can present a high
bleeding risk in patients with existing hemostatic defects, but typically does not result in clinically
TABLE 11–17 Laboratory Evaluation for Hemostatic Defects in Uremia

PT and PTT Normal
Platelet count May be decreased, but it is rarely <80,000/μL; hemodialysis can exacerbate
the thrombocytopenia, but the function of the platelets may improve
Platelet aggregation studies No typical pattern of responses to platelet agonists; decreased response to
ADP, collagen, and epinephrine often observed
ADP, adenosine diphosphate; PT, prothrombin time; PTT, partial thromboplastin time.
TABLE 11–18 Drug-induced Qualitative Platelet Dysfunction

Platelet count The platelet count is necessary to identify an underlying thrombocytopenia, if

one exists, which is aggravated by a drug effect
PT and PTT, von Willebrand Abnormal only if there is an underlying coagulation factor abnormality
factor antigen, and ristocetin
cofactor
Platelet aggregation studies Abnormal platelet aggregation may be observed in patients exposed to
certain drugs in vivo, especially to weak platelet agonists such as epinephrine;
however, the presence of abnormal aggregation does not correlate well with
the risk of bleeding
significant bleeding in normal individuals. When hemorrhage does occur, there is usually an
underlying hemostatic disorder affecting either the platelets or coagulation factors, or an ana-
tomic lesion, such as an ulcer, that predisposes the patient to bleeding.
Commonly encountered coagulopathies that place patients at risk for bleeding when there is
a superimposed drug-induced platelet defect include vWD, thrombocytopenia of any cause, and
anticoagulation therapy. Hemorrhagic manifestations can include petechiae and purpura, ecchy-
moses, mucosal membrane bleeding, hematuria, epistaxis, and oozing from intravenous access
sites and surgical incisions.
Diagnosis
Laboratory tests are of little value in predicting the clinical significance of drug-induced plate-
let defects. They can confirm the presence of abnormal platelet function, but cannot assess the
risk of bleeding. Furthermore, laboratory abnormalities in platelet function are not specific for a
particular drug. See Table 11–18 for information on the laboratory evaluation for drug-induced
platelet defects.
THROMBOTIC DISORDERS
In this chapter, the disorders associated with thrombosis (Figure 11–5) are grouped into those The disorders associated
with a relatively higher prevalence and those with a relatively lower prevalence. Among those with with thrombosis are
a higher prevalence is activated protein C resistance, which is produced by the Factor V Leiden grouped into those
mutation. This mutation is present in 3% to 5% of Caucasian populations. Other thrombotic with a relatively higher
disorders with a high prevalence are the prothrombin G20210A mutation, and the antiphospho- prevalence and those
lipid antibody syndrome (an acquired disorder). The thrombotic disorders with a lower preva- with a relatively lower
prevalence. Among those
lence include protein C deficiency, protein S deficiency, and antithrombin deficiency. Elevated
with a higher prevalence
plasma homocysteine levels may also increase the risk for thrombosis. Plasminogen deficiency is
are activated protein
also rare, and its association with thrombosis is controversial. Two other rare conditions, dysfi-
C resistance, which is
brinogenemia of certain types and essential thrombocythemia, can produce either thrombosis or produced by the Factor
bleeding. Also rare are TTP and HUS. V Leiden mutation, the
prothrombin G20210A
mutation, and the
Hypercoagulable States antiphospholipid
Description antibody syndrome
Hypercoagulable states are associated with an increased risk for thrombosis (Table 11–19). There (an acquired disorder).
are both hereditary and acquired hypercoagulable states. Hereditary forms may arise from a
quantitative or qualitative deficiency of a regulatory anticoagulant protein, such as protein C, pro-
tein S, or antithrombin (see Figures 11–2 and 11–3). Activated protein C resistance is caused by a
mutation in the Factor V molecule (nearly always the Factor V Leiden mutation), which prevents
activated protein C-mediated inactivation of Factor Va. The prothrombin G20210A mutation
is prevalent in Caucasian populations, similar to Factor V Leiden. Prothrombin G20210A is a
TABLE 11–19 Laboratory Evaluation for Hypercoagulable States

Incidence in General Relevant Laboratory
Hypercoagulable State Population Site of Thrombosis Test Results Comments
Inherited
Activated protein C 3%-5% in Caucasians Predominantly venous Positive activated Uncommon in those
resistance (nearly always protein C resistance of African and Asian descent
associated with the test; DNA test positive
presence of the Factor V for Factor V Leiden
Leiden mutation)
Prothrombin G20210A 1.5%-3% in Caucasians Predominantly venous DNA test positive Uncommon in those
mutation for prothrombin of African and Asian descent
G20210A
Hyperhomocysteinemia Markedly high values Venous and arterial; often At least moderately It has been shown that
(especially congenital are extremely rare; mild with atherosclerosis elevated homocysteine vitamins do not decrease
forms with extremely elevations are common thrombotic risk
high values); can also
be acquired
Protein C deficiency 0.2%-0.4% Predominantly venous Low functional and Risk of warfarin-
(congenital deficiency (if type I deficiency) induced skin necrosis
only) antigenic protein C if anticoagulation is
initiated with warfarin
in the absence of
heparin
Protein S deficiencya 0.2%-0.4% Predominantly venous Low functional and Estrogen, pregnancy,
(congenital deficiency (if type I deficiency) and oral contraceptives
only) antigenic protein S cause acquired
decreases, as do
acute-phase
reactions
Antithrombin deficiencya 0.01%-0.02% Predominantly venous Low functional and Heparin use can cause an
(congenital deficiency (if type I deficiency) acquired deficiency
only) antigenic antithrombin
Acquired
Antiphospholipid antibody 1%-5% in the general Venous and arterial Positive test results for To make a diagnosis of
(APA) (the presence of a population; much lupus anticoagulant antiphospholipid syndrome,
lupus anticoagulant, an higher incidence in and/or anticardiolipin test results for the lupus
anticardiolipin antibody, groups with certain antibody, and/or anti- anticoagulant, anticardiolipin
and/or anti-beta-2 underlying conditions, beta-2 glycoprotein I antibody, and/or anti-
glycoprotein I antibody) especially systemic lupus antibody beta-2 glycoprotein I
erythematosus; higher antibody must be positive
incidence with age on 2 separate occasions
12 weeks apart, in the setting
of thrombosis, or specific
pregnancy complications,
occurring within 5 years of a
positive test
Selected other acquired predisposing conditions for thrombosis
For venous thromboembolism: postoperative state, immobility, trauma, obesity, congestive heart failure, pregnancy and postpartum state, estrogen
and progesterone use, nephrotic syndrome, L-asparaginase therapy, infection, prolonged travel, dehydration, smoking, and malignancy
For arterial thromboembolism: atherosclerosis, damaged endothelium, bypass grafts, cardiac emboli (from atrial fibrillation, mitral stenosis, or mural
thrombus following myocardial infarction), and arteritis
For both venous and arterial thromboembolism: disseminated intravascular coagulation, malignancy, myeloproliferative disorders, systemic lupus
erythematosus, paroxysmal nocturnal hemoglobinuria, and heparin-induced thrombocytopenia
a
If both protein C and protein S are decreased, vitamin K deficiency or warfarin intake should be considered, especially if the prothrombin time is prolonged;
if protein C, protein S, and antithrombin are all decreased, decreased synthesis of these proteins from liver disease, or recent/active clotting with consumption
of the factors, may be the explanation.
mutation in the promoter of the prothrombin gene, causing increased synthesis of prothrombin
(Factor II). Hyperhomocysteinemia, a disorder in which there is an abnormally high level of
plasma homocysteine, can be hereditary or acquired. Hyperhomocysteinemic individuals are at
increased risk for coronary artery disease and deep venous thrombosis. However, studies have
not yet shown that reducing slightly or moderately elevated homocysteine with vitamin therapy
reduces the thrombotic risk. A block in the pathway at any 1 of the several steps results in the
accumulation of homocysteine. When the homocysteine level is manyfold higher than the upper
limit of normal, this very high value may contribute to the damaging effects on the blood ves-
sel wall. Acquired hypercoagulable states arise from a diverse array of clinical conditions. They
include malignancy, immobilization, surgery, trauma, obesity, smoking, infection, prolonged
travel, and the use of oral contraceptives, estrogen replacement therapy, and progesterone, among
many others. An acquired hypercoagulable state for which specific coagulation testing is available
is the antiphospholipid antibody syndrome.
The presence of 1 hypercoagulable condition alone is not usually sufficient to initiate throm-
bosis. The presence of a second (or more), superimposed hypercoagulable condition (often called
a “second hit”) appears to be required to provoke a thrombotic event. For example, a person
with activated protein C resistance may not experience a thrombotic event until suffering major
trauma as a “second hit.”
Diagnosis
Laboratory testing for hypercoagulable states is most often performed for patients present- Laboratory testing for
ing with a personal or family history of thrombosis. The laboratory evaluation for hereditary hypercoagulable states
hypercoagulable states is best performed as a panel of test results. The most common disorders is most often performed
are activated protein C resistance (caused nearly always by Factor V Leiden) and prothrombin for patients presenting
G20210A, and the less common are deficiencies of protein C, protein S, and antithrombin. Fre- with a personal or family
quently, antiphospholipid antibody testing is performed in conjunction with the tests for heredi- history of thrombosis.
tary hypercoagulable disorders. If all of these tests are negative and the clinical suspicion for a The laboratory
evaluation for hereditary
congenital hypercoagulable state remains high, additional testing for the more rare hypercoagu-
hypercoagulable states is
lable disorders can be performed. There are many acquired conditions or treatments that reduce
best performed as a panel
the level of protein C, protein S, and antithrombin in the test panel for hypercoagulability, but of test results.
despite this, the risk for thrombosis is not significantly increased on that basis. For example, war-
farin reduces the levels of protein C and protein S but reduces thrombotic risk. Antithrombin is
lowered by heparin therapy. Pregnancy, oral contraceptives, and estrogen therapy can all decrease
the activity of protein S, although they can induce a hypercoagulable state by other mechanisms.
Active clot formation, liver dysfunction, or DIC can lower protein C, protein S, and antithrombin.
In situations where such a confounding variable exists that alters the level of protein C, protein S,
or antithrombin, the tests should be repeated (if possible) when the variable is no longer present
to obtain the patient’s true baseline values. This should allow determination of whether a heritable
deficiency of any of these 3 proteins truly exists:
• Activated protein C resistance: Usually, the first assay performed is a functional assay for
activated protein C resistance. If the result is abnormal, genetic analysis is performed,
to confirm whether Factor V Leiden is present in the heterozygous state, present in the
homozygous state, or absent.
• Prothrombin G20210A: This mutation is identified by genetic analysis that can specifically
identify heterozygous and homozygous states.
• Hyperhomocysteinemia: Plasma homocysteine levels are measured by a variety of
automated methods.
• Protein C, protein S, and antithrombin deficiencies: Individual functional assays to measure
the activity of these endogenous anticoagulant proteins detect both qualitative (normal
number of abnormally functioning molecules) and quantitative (low number of normally
functioning molecules) deficiencies. In contrast, an antigenic (immunologic) assay, which
measures only the quantity of protein present, can detect quantitative but not qualitative
deficiencies. Therefore, the first assay performed should be a functional assay. If the result
is abnormal, an antigenic assay can be performed to determine if the cause of the decreased
activity is a quantitative or qualitative deficiency of the protein.
• LA (an antiphospholipid antibody): A variety of tests can be used to detect an LA. This
antibody interferes with the cofactor action of phospholipid in the coagulation cascade
in laboratory assays only (see the section “Antiphospholipid Antibodies: The Lupus
Anticoagulant, Anticardiolipin Antibodies, and Beta-2 Glycoprotein I Antibodies” [Beta-2
GP I antibodies]). Various phospholipid-dependent coagulation test times, especially PTT-
based or dilute Russell viper venom time (DRVVT) assays, can be used. The DRVVT is the
clotting time obtained using Russell viper venom, which contains a Factor X activator.
• Anticardiolipin antibody or beta-2 GP I antibody (both are antiphospholipid
antibodies): These antiphospholipid antibodies may or may not be associated with
the presence of an LA (see the section “Antiphospholipid Antibodies: The Lupus
Anticoagulant, Anticardiolipin Antibodies, and Beta-2 Glycoprotein I Antibodies”),
and they are detected by enzyme-linked immunoassays.
See Table 11–19 for characteristics of the hypercoagulable states.
Antiphospholipid Antibodies: The Lupus Anticoagulant,

Anticardiolipin Antibodies, and Beta-2 Glycoprotein I Antibodies
Description
Antiphospholipid antibodies recognize specific phospholipid–protein complexes rather than
phospholipid alone. They can be immunoglobulin type IgG or IgM, or, less commonly, IgA. The
LA is an immunoglobulin that can interfere with phospholipid-dependent coagulation reactions
in laboratory assays without inhibiting the activity of any specific coagulation factor. It targets
phospholipids bound to prothrombin, beta-2 GP I, protein C, protein S, or other proteins bound
to phospholipids. Anticardiolipin antibodies are another type of antiphospholipid antibody,
which target beta-2 GP I bound to a particular phospholipid, cardiolipin; these can be detected
by anticardiolipin antibody immunoassays. Anti-beta-2 GP I antibodies also target beta-2 GP I.
An antiphospholipid antibody may occur in apparently healthy individuals with no detect-
able illness. It also occurs in patients with a variety of clinical conditions or disorders including:
• Systemic lupus erythematosus and other autoimmune disorders
• Malignancy
• Following ingestion of selected drugs (procainamide, quinidine, phenytoin, chlorpromazine,
valproic acid, amoxicillin, augmentin, hydralazine, streptomycin, and propranolol have all
been reported to induce an LA)
• Infectious diseases—bacterial (including spirochetal and mycobacterial), viral, fungal, and
protozoal infections
• Following vaccination
A lupus anticoagulant is Estimates of prevalence have been highly variable because results are completely dependent
the most common cause on the test(s) used for detection of antiphospholipid antibodies, and some methods are more sen-
of a prolonged PTT that sitive than others. Approximately 2% of patients with a prolonged PTT will have an LA as the cause
remains prolonged in a of the prolongation. LA is the most common cause of a prolonged PTT that remains prolonged in
PTT mixing study (a PTT a PTT mixing study (a PTT performed on a sample of mixed patient and normal plasma). It is esti-
performed on a sample of mated that 1% to 5% of the general population has an antiphospholipid antibody. The frequency
mixed patient and normal of antiphospholipid antibody in systemic lupus erythematosus patients is in the 30% to 40% range.
plasma). Antiphospholipid antibodies due to infections are typically transient and asymptomatic.
Although the LA acts as an anticoagulant in vitro, it does not appear to be associated
with hemorrhage, even with surgical challenge. Rare cases of bleeding in patients with the
LA can almost always be attributed to specific abnormalities in hemostasis that happen to be
present along with the LA. Decreased prothrombin (Factor II) is occasionally found with the
LA. The LA can bind directly to prothrombin, but typically the LA does not neutralize the
procoagulant activity of prothrombin even when it does bind to it. In an occasional patient,
however, antibody binding does reduce the prothrombin concentration by accelerated clear-
ance of prothrombin/antiprothrombin complexes, and these patients can have hemorrhagic
complications. Concomitant thrombocytopenia is not infrequently found in patients with the
LA, and this also may be a cause for hemorrhage.
Antiphospholipid antibodies are associated with an increased risk for venous and arterial
thrombosis. The role of the antiphospholipid antibody in thrombosis is not clear, although several
mechanisms for thrombosis have been proposed. The incidence of clinically apparent thrombo-
embolism in patients with the LA, with or without systemic lupus erythematosus, is difficult to
determine because of the variety of tests used to detect the LA. However, data suggest that the per-
centage of patients with the LA who will develop thrombosis is 1% per year if there is no history
of thrombosis and 5.5% per year if there has been at least 1 prior thrombotic event. High titers
of anticardiolipin or anti-beta-2 GP I antibodies present a higher risk for complications than low
titers, and IgG is thought to be higher risk than IgM or IgA. There is a greater risk for thrombosis
if more than 1 of the 3 antiphospholipid antibody tests (LA, anticardiolipin antibodies, and anti-
beta-2 GP I antibodies) are abnormal.
Recurrent spontaneous abortion has been reported to be increased in patients with antiphos-
pholipid antibodies. There is evidence suggesting that thrombosis and infarction in the placenta
mediate antiphospholipid antibody-associated spontaneous abortion in a significant number of
women experiencing recurrent fetal loss or premature birth.
Diagnosis
There are no “gold standard” tests that unequivocally establish the presence of antiphospholipid
antibodies:
• For the LA: A prolonged PTT and a PTT mixing study that does not correct into the normal
range are clues to the presence of the LA, although some PTT reagents are not sensitive to
the LA. Therefore, it should be noted that the routine PTT is not an appropriate screening
test for the LA. A PTT- or DRVVT-based test with a reduced amount of phospholipid
can be used as a screening test for the LA. If it is prolonged, a 1:1 mixing study and a
confirmatory assay should be performed. In the presence of an LA, the PTT or DRVVT
usually remains prolonged when equal portions of patient plasma and normal plasma are
mixed. Confirmatory assays demonstrate that the clotting time shortens toward normal
when excess phospholipid is added, overcoming the LA effect. The PT is not typically
increased by the LA, unless the patient has an associated hypoprothrombinemia or the
thromboplastin used in the PT is one that is particularly sensitive to inhibition by the LA.
• For anticardiolipin or anti-beta-2 GP I antibodies: An enzyme-linked immunosorbent assay
(ELISA) is used that quantitates IgG and IgM antibody levels in arbitrary units. IgA is also
measured in some kits.
Patients with antiphospholipid antibodies may have a false-positive serologic test result for
syphilis (such as Venereal Disease Research Laboratories [VDRL] and rapid plasma reagin [RPR]).
See Table 11–19 for information on the laboratory diagnosis of antiphospholipid antibodies.
Thrombotic Thrombocytopenic Purpura Thrombotic

thrombocytopenic
Description purpura is a clinical
TTP is a clinical syndrome that is characterized by a triad (1-3 below, more commonly) or pentad syndrome that is
(1-5 below, less commonly) of signs and symptoms: characterized by a triad
(more commonly) or
1. Thrombocytopenia with generalized purpura, and mucous membrane bleeding
pentad (less commonly)
2. Hemolytic anemia (microangiopathic) sufficient to cause jaundice or pallor
of signs and symptoms.
3. Neurologic abnormalities, which may include fluctuating weakness, dysphagia, headache,
dementia/behavioral changes, obtundation, seizures, diplopia, paresthesias, and coma
4. Fever
5. Renal dysfunction, which may include hematuria, proteinuria, or renal insufficiency
The characteristic, but not pathognomonic, pathology includes platelet and fibrin “hya-
line” thrombi in the small arteries, arterioles, and capillaries in a widespread organ distribution.
Organ ischemia and infarction that arise from these thrombi are thought to give rise to the
observed fever and organ dysfunction. The etiology for TTP has been shown to be a deficiency
of vWF-cleaving protease. Nonfamilial cases of TTP are a result of an inhibitor to the vWF-
cleaving protease; the familial form of TTP is apparently caused by a constitutional deficiency of
TABLE 11–20 Laboratory Evaluation for Thrombotic Thrombocytopenic Purpura

Activity of von Willebrand factor- A low value for this enzyme activity is the diagnostic hallmark for the
cleaving protease activity disease, but the assay may not be available for rapid diagnosis of TTP.
If the value is low, tests to detect an inhibitor to the protease can
be performed
PT, PTT, and fibrinogen levels Usually normal
Fibrin degradation products or Normal or slightly elevated

D-dimer
Hemoglobin/hematocrit Mild-to-moderate decrease in most cases; hemorrhage and hemolysis

can result in severe anemia in some patients
Haptoglobin Low as a reflection of intravascular hemolysis
Platelet count Decreased, often in the range of 10,000-50,000/μL
Peripheral blood smear Shows schistocytes, nucleated RBCs, and decreased platelet number
Direct and indirect antiglobulin tests Negative, ruling out an immune hemolytic anemia
Indirect bilirubin Mild-to-moderate elevation
Serum lactate dehydrogenase (LDH) Elevated, correlating with the severity of hemolysis and possibly
tissue damage from ischemia
WBC count and differential Shows a mild leukocytosis with a left shift
Urinalysis Characterized by mild-to-moderate proteinuria and hematuria

(without casts)
BUN and creatinine May or may not be elevated, depending on the presence of
renal impairment
BUN, blood urea nitrogen; PT, prothrombin time; PTT, partial thromboplastin time.
the protease. The unusually large forms of vWF in the plasma of patients with TTP promote the
aggregation of platelets in vivo, which accounts for most of the clinical findings.
TTP can occur at any age but is most common between the ages of 20 and 50 years. Peak
incidence is in the third decade. There is a female to male ratio of 3:2. TTP usually occurs as an
acute, fulminant disease, but may also occur in a chronic form or in an acute relapsing form. The
acute and acute relapsing types are often preceded by a viral prodrome. Nonspecific signs such as
malaise, weakness, fatigue, and anorexia may predominate at first, until the above triad or pentad
develops in days to weeks. The chronic type usually pursues an indolent, low-grade course, with
ongoing disease activity for months.
Nonspecific manifestations in other organ systems resulting from ischemia may include:
• Cardiac: conduction defects, sudden death, and heart failure
• Pulmonary: lung infiltrates and acute respiratory failure
Hemolytic–uremic • Gastrointestinal: abdominal pain due to visceral ischemia, pancreatitis, and gastrointestinal
syndrome is a clinical mucosal hemorrhage
syndrome with
presentation and Diagnosis
manifestations similar to
See Table 11–20 for information on the laboratory evaluation for TTP.
TTP. Despite the clinical
similarity, evidence has
shown that the low levels Hemolytic–Uremic Syndrome
of vWF-cleaving protease
Description
found in TTP are not
found in HUS. HUS is a clinical syndrome with presentation and manifestations similar to TTP. Despite the clin-
ical similarity, evidence has shown that the low levels of vWF-cleaving protease found in TTP are
not found in HUS. Thus, the pathogenesis of these 2 disorders is apparently completely different.
HUS is characterized by fever, microangiopathic hemolytic anemia, thrombocytopenia, and renal
dysfunction. It differs clinically from TTP in the following ways: neurologic symptoms are less
TABLE 11–21 Laboratory Evaluation for Hemolytic–Uremic Syndrome

PT, PTT, and fibrinogen Normal
Fibrin degradation products or Absent or minimally increased

D-dimer
Hemoglobin/hematocrit Decreased with microangiopathic changes on the peripheral blood

smear (nucleated RBCs, schistocytes)
Platelet count Mild-to-moderate decrease
Urinalysis Hematuria, proteinuria, and red cell casts
Creatinine Elevated
Lactate dehydrogenase (LDH) Elevated
Indirect bilirubin Elevated
Haptoglobin Low
Direct antiglobulin test (DAT) Negative

E. coli O157:H7 Commonly positive
pronounced or absent in HUS; renal function is usually more impaired in HUS than in TTP;
HUS occurs in a younger population than TTP with a peak incidence between 6 months and 4
years, with males and females equally affected; HUS is a more common entity than TTP; as with
TTP, hyaline thrombi may be found, but in most cases they tend to be confined to the glomerular
capillaries and afferent arterioles.
HUS occasionally occurs in adults, and is often distinguished from childhood HUS because
of its strong association in adults with obstetrical complications such as eclampsia. The prognosis
is worse in adults than in affected children, with an adult mortality as high as 60%.
Acute HUS occurs in nonfamilial and familial forms, which may have different causes.
Nonfamilial forms are most often associated with a diarrheal illness caused by a Shiga-toxin-
producing E. coli (in particular, E. coli O157:H7). Familial forms have been linked to abnormalities
in complement factor H and factor I. The majority of childhood cases resolve without sequelae,
if children with acute renal failure receive dialysis when necessary. The prognosis depends on the
duration of renal failure and the severity of the neurologic disturbance. Renal function returns to
normal in approximately 90% of childhood cases.
Diagnosis
See Table 11–21 for information on the laboratory evaluation for HUS.
Anticoagulant Therapies
Thrombosis can be treated and/or prevented with anticoagulant therapies, which are therapies
that inhibit the coagulation cascade, thereby inhibiting fibrin clot formation. Since too little anti-
coagulation might allow new thrombosis to occur while too much anticoagulant can cause bleed-
ing, laboratory tests can be used to determine how much anticoagulation is present in the patient,
so that the dose can be adjusted if needed. For decades, heparin and warfarin were the only anti-
coagulants available for use. Heparin is given intravenously or subcutaneously, and warfarin is
taken orally. As described previously, heparin inhibits multiple coagulation factors, and the effect
of this inhibition is that heparin prolongs the PTT. Most PT reagents contain a heparin neutral-
izer that largely prevents significant PT prolongations for patients on heparin. Warfarin decreases
the synthesis of active forms of Factors II, VII, IX, and X, which prolongs the PT. Different PT
methods can demonstrate different degrees of prolongation with warfarin. Therefore, a formula
is used to convert the PT into a more standardized number that takes into account the relative
sensitivity of the PT reagent. This calculation is called the international normalized ratio (INR),
TABLE 11–22 Laboratory Monitoring of Anticoagulant Therapies

Anticoagulant Mechanism Tests to Monitor Anticoagulation
Warfarin Decreases active forms of Factors II, VII, IX, X INR
Heparin Inhibits Factors IIa, Xa, IXa, XIa, Xa, XIIa PTT a
LMWH Inhibits Factor Xa Anti-Factor Xa
Fondaparinux Inhibits Factor Xa Anti-Factor Xa
Rivaroxaban Inhibits Factor Xa Anti-Factor Xa
Apixaban Inhibits Factor Xa Anti-Factor Xa
Argatroban Inhibits Factor IIa PTT

Dabigatran Inhibits Factor IIa Dilute thrombin time
Bivalirudin Inhibits Factor IIa ACT for cardiac catheterization

INR, international normalized ratio; PTT, partial thromboplastin time; ACT, activated clotting time.
a
For high doses during cardiac catheterization, the ACT is used.
and it is meant to provide similar results no matter what method is in use by the laboratory. To be
sure the correct amount of anticoagulation is present, heparin is closely monitored with the PTT,
and warfarin is closely monitored with INR measurements.
Newer anticoagulants have emerged that do not routinely need monitoring with laboratory
tests. Nevertheless, laboratory monitoring may still be occasionally indicated. Low-molecular-
weight heparin, fondaparinux, rivaroxaban and apixaban inhibit Factor Xa. Therefore, these anti-
coagulants can be monitored with anti-Factor Xa assays. These are assays that can measure the
concentration of the anticoagulant based on how much inhibition of Factor Xa is detected. Direct
thrombin inhibitors (argatroban, bivalirudin, and dabigatran) inhibit thrombin, which prolongs
the PTT and to a lesser extent the PT. A dilute thrombin time assay has been shown to have a lin-
ear relationship to therapeutic doses of dabigatran. The PTT can be useful to monitor argatroban.
Bivalirudin is specifically approved for use during percutaneous coronary intervention (PCI), a
cardiac catheterization procedure that requires anticoagulation that is monitored with a rapid,
bedside whole blood clotting time test called the activated clotting time (ACT). If a patient has
PCI with bivalirudin, the ACT is used.
Only warfarin, rivaroxaban, dabigatran and apixaban are administered orally. The other anti-
coagulants are administered parenterally.
See Table 11–22 for a summary of the mechanisms of action and the laboratory tests to mea-
sure the anticoagulation effect of these various anticoagulant therapies.
Antiplatelet Therapies
Patients with arterial thrombosis, in particular myocardial infarction or ischemic stroke, are usu-
ally treated with antiplatelet therapy, to inhibit platelet activation. Unlike many anticoagulant
therapies, laboratory testing is not yet routinely used to monitor antiplatelet therapy. Studies are
ongoing to determine if laboratory monitoring of platelet therapy is beneficial. A variety of plate-
let-inhibiting medications are available. Aspirin irreversibly inhibits cyclooxygenase in platelets,
which prevents the generation of thromboxane A2 from arachidonic acid. This inhibits platelet
aggregation because thromboxane A2 triggers the release of platelet granules and activates other
platelets. Several GP IIb/IIIa inhibitors are available (abciximab, eptifibatide, or tirofiban). These
agents inhibit platelet aggregation because GP IIb/IIIa on the platelet surface mediates platelet
aggregation. Clopidogrel, prasugrel, and ticagrelor all inhibit an ADP receptor on the platelet sur-
face, which ultimately inhibits activation of platelet GP IIb/IIIa. Dipyridamole inhibits platelets
through multiple mechanisms.
A portion of this chapter, primarily the material in the section “Introduction to Hemostasis,” was
adapted with permission with modifications from Laposata M, Connor AM, Hicks, DG, Phillips DK.
The Clinical Hemostasis Handbook. Chicago: Year Book Medical Publishers; 1989.
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C H A P T E R
Transfusion Medicine
Christopher P. Stowell and Jacqueline J. Haas 12
LEARNING OBJECTIVES
1. Understand the process of blood collection and the preparation of blood
components and plasma derivatives.
2. Learn which tests must be performed to assure safe transfusion.
3. Learn the specific indications for transfusion of individual blood components
and alternatives to allogeneic transfusion.
4. Understand the clinical complications that may arise after transfusion
of blood components.
5. Learn about the collection and use of hematopoietic progenitor cells.
6. Learn the process of apheresis and its clinical indications.
CHAPTER OUTLINE
Collection of Blood and Preparation Molecular Techniques in
of Blood Components 292 Immunohematology 302
Blood Collection 292 Indications for Transfusion 303
Component Preparation 293 Red Blood Cells 303
Testing of Donated Blood 296 Platelets 304
Infectious Disease Testing 296 Fresh Frozen Plasma 304
Compatibility Testing 297 Cryoprecipitate 304
Pretransfusion Testing to Assess Donor/ Complications of Blood Transfusion 304
Recipient Compatibility for Blood Immunologic Reactions 304
Components Containing RBCs 297 Nonimmunologic Reactions 307
ABO Grouping 298 Transfusion Reaction Workup 309
Rh Typing 299 Alternatives to Allogeneic Transfusion 310
The Antibody Screen and the Indirect Cellular Therapies 311
Antiglobulin Assay Used to Detect Therapeutic Apheresis 312
Antibodies 299 Indications for Therapeutic Apheresis 312
The RBC Crossmatch 301 Plasmapheresis 314
Direct Antiglobulin Test (Formerly Cytapheresis 314
Direct Coombs Test) 301 Erythrocytapheresis (RBC Exchange) 314
Compatibility Testing for Other Blood Photopheresis 314
Components That Do Not Contain RBCs 302
T
ransfusion medicine is the field of medicine that encompasses blood banking (the col-
lection, preparation, testing, and storage of blood components and plasma derivatives)
as well as the therapeutic uses of blood components, plasma derivatives, and apheresis
technology. It also includes the collection, storage, and use of hematopoietic and other blood-
derived cells. An overview of the steps from collection of the blood to transfusion of its com-
ponents is shown in Figure 12–1. Briefly (with more complete descriptions to follow), blood is
291
292 CHAPTER 12 Transfusion Medicine
Identification of
Testing of blood
Blood component appropriate
Blood collection for infectious
preparation components
disease
for treatment
After correct
If necessary,
identification Evaluation of
treatment of
of the patient the patient
Assessment of component
and the product for complications
donor-recipient prior to transfusion
intended for of transfusion
compatibility to minimize
transfusion–– and response to
adverse
transfusion transfusion
effects
of the patient
FIGURE 12–1 An overview of blood collection, processing, and transfusion.
collected as whole blood or by apheresis from screened, volunteer donors, and samples of the
blood are tested for infectious diseases and to determine the blood type. Whole blood may be
fractionated into packed red blood cells (RBCs), platelets, and a plasma product. Alternatively,
all 3 components can be collected by apheresis. Plasma can be further processed to provide
albumin, clotting factor concentrates, and immunoglobulin preparations. The transfusion of
blood components requires testing to be done to establish compatibility between the product
Whole blood may be and the intended recipient. Blood components may also be treated to reduce complications
fractionated into packed of transfusion (eg, remove leukocytes to prevent febrile reactions). As complex, biologically
red blood cells (RBCs), derived therapeutic agents, blood components and derivatives are responsible for a variety
platelets, and a plasma of potential untoward effects that must be evaluated and managed. The entire process, from
product. blood collection to transfusion and posttransfusion evaluation, is described in this chapter
(see Figure 12–1).
COLLECTION OF BLOOD AND PREPARATION

OF BLOOD COMPONENTS
Blood Collection
The cornerstone of a safe blood supply is the volunteer blood donor who is motivated by altruism.
In the past, the use of paid donors was associated with increased levels of transfusion-transmitted
hepatitis. Concerns remain about the impact of significant financial incentives on the frank dis-
closure of health problems or high-risk behaviors that might disqualify a potential blood donor.
In the United States, virtually all of the blood is collected from volunteer donors. Regional blood
centers collect and distribute more than 90% of the US blood supply while hospital blood banks
collect the remainder. The Food and Drug Administration (FDA) Center for Biologics Evaluation
and Research regulates all aspects of blood collection and processing, but most blood banks and
donor centers are also accredited, on a voluntary basis, by the American Association of Blood
Banks (AABB). Blood donors are screened for behaviors or medical conditions that might make
blood donation unsafe for them (eg, anemia, coronary artery insufficiency) or the donated blood
hazardous for the transfusion recipient (eg, exposure to viral hepatitis, use of a teratogenic medi-
cation). The AABB has developed, and the FDA has sanctioned, a Uniform Donor Health Ques-
tionnaire that is in wide use in the United States and reflects the consistency of donor criteria
throughout the country. To qualify for blood donation, the prospective donor must also pass a
basic physical screening that includes temperature, blood pressure, pulse, and examination of the
arms for signs of intravenous drug use, and have a hemoglobin level of at least 12.5 g/dL from a
CHAPTER 12 Transfusion Medicine 293
finger-stick or venous blood sample. The collecting agency must check its records to be sure that
the donor has not previously been disqualified from donating.
In the process of blood collection, the venipuncture site is disinfected and a needle, which is
connected to a collecting set, is inserted into a vein in the arm. The collecting set includes a pri-
mary collection bag and several integrally connected satellite bags that are used to make compo-
nents. The primary collection bag contains a solution that includes an anticoagulant (citrate) and
a variety of substances, such as phosphate, adenine, and dextrose, which improve the recovery
The typical donation is
of the RBCs when transfused and permit their storage in the refrigerator for 35 to 42 days. The
approximately 450 to
typical donation is approximately 450 to 500 mL of whole blood, but may not exceed 10.5 mL/kg, 500 mL of whole blood.
and must be collected in no more than 10 minutes if a unit of platelets is to be made. This volume This volume of blood
of blood represents approximately 10% of the total blood volume of a donor weighing 70 kg, represents approximately
and its loss is well tolerated by healthy individuals. Samples of blood for serologic and infectious 10% of the total blood
disease testing are also obtained at the time of donation, often by collecting the first 15 to 25 mL volume of a donor
of blood drawn, which has the advantage of diverting blood most likely to be contaminated with weighing 70 kg, and its
skin bacteria away from the collection bag. Once the collection is complete, the needle is with- loss is well tolerated by
drawn from the donor’s arm, and the tubing connecting the needle to the primary collection bag healthy individuals.
is heat-sealed off.
Component Preparation
Almost all of the whole blood collected is separated into its components—RBCs, platelets, and
plasma—in order to be able to store each under optimal conditions. This separation process entails
2 centrifugation steps and relies on the system of integrally connected satellite bags to carry out all
of the preparation steps in a closed, aseptic environment (see Table 12–1 for a description of blood
components, and Chapter 2 for a diagram of blood component preparation). In the procedure
used in the United States, RBCs are separated from platelet-rich plasma by the first, relatively low
g force, centrifugation step. The platelet-rich plasma is expressed from the primary collection bag
into one of the satellite bags that is heat-sealed off from the packed RBCs in the primary collection
bag. The packed RBCs remaining in the primary collection bag may be stored for up to 35 days at
1°C to 6°C (CPDA-1 RBCs). In some collection systems, additional additive–preservative solution
from another satellite bag may be added to the packed RBC, creating a product with a lower
hematocrit (but identical RBC mass) and an additional week of storage (42 days).
The platelet-rich plasma, which was expressed off the packed RBC after the first spin, is then
separated into platelets and plasma by a second, higher g force, centrifugation step. The platelet-
poor plasma is expressed into another satellite bag after this second centrifugation step and is
usually frozen within 8 hours of collection (as fresh frozen plasma [FFP]) or within 24 hours
of collection (24-hour plasma). The platelet pellet that remains is suspended in 40 to 60 mL of
plasma and is called a platelet concentrate or whole blood-derived platelets. The term “random
donor platelets,” although commonly used, is inaccurate. Platelets are stored at 20°C to 24°C for
up to 5 days, whereas the various plasma-derived components are stored frozen (≤−18°C for
1 year; ≤−65°C for 7 years). Since 1 transfusion dose for an adult patient consists of 4 to 10 units
of platelet concentrates, it is common to use commercially available closed systems to pool and
leukoreduce them.
FFP can be used to prepare another useful component, called cryoprecipitated antihemo-
philic factor (or “cryoprecipitate”). When FFP is thawed at 1°C to 6°C, a precipitate forms. Most
of the plasma can be expressed into a satellite bag, leaving behind this cryoprecipitate that is sus-
pended in 10 to 20 mL of plasma, refrozen, and then stored (≤−18°C for 1 year). Cryoprecipitate
contains about half of the Factor VIII, von Willebrand factor, Factor XIII, and fibrinogen that was
present in the original unit of plasma, but in a much smaller volume. The plasma remaining after
the cryoprecipitate has been removed may also be refrozen. “Plasma–cryoprecipitate reduced”
(or cryo-poor plasma) may be used as the starting material (source plasma) for the preparation
of plasma derivatives such as albumin and immunoglobulins, and occasionally for replacement
during plasmapheresis for patients with thrombotic thrombocytopenic purpura (see Table 12–2).
Thus, each unit of whole blood can be separated into a unit of packed RBCs, a platelet concen-
trate, and either FFP or cryoprecipitate plus some other plasma product. The different plasma
products (absent cryoprecipitate) may all be used as source plasma for the preparation of deriva-
tives, such as albumin or immunoglobulin.
TABLE 12–1 Blood Component Descriptions and Indications

Category Description of Product Major Indications Actions Precautions
Packed RBCs Packed RBCs are the Packed RBCs are used for Packed RBCs provide RBCs must be ABO-
product of a centrifugal treatment of a symptomatic RBC mass and increase and Rh-compatible,
separation of red cells anemia; this component oxygen-carrying and crossmatched
from plasma; this may also be used for capacity in the blood
component has a exchange transfusion
hematocrit of 55%-80% when treating sickle cell
crisis or hemolytic disease
of the newborn
RBCs, leukoreduced Packed RBCs may be The indications for this Packed RBCs provide RBCs must be ABO-
modified by removal of product are the same as for RBC mass and increase and Rh-compatible,
leukocytes by filtration or packed RBCs; leukoreduced oxygen-carrying and crossmatched
washing; washing RBCs is RBCs are used for individuals capacity in the blood
less effective at removing who have experienced febrile
leukocytes than filtration reactions due to passenger
techniques WBCs in a blood component;
they are also used to prevent
alloimmunization in a
patient who may require
multiple platelet transfusions,
or to prevent cytomegalovirus
infection in a susceptible
patient
Fresh frozen Plasma that is separated Because FFP contains FFP restores plasma This component
plasma (FFP) from the cellular significant levels of all the proteins, particularly should be ABO-
components and frozen plasma coagulation factors, coagulation factors, compatible with the
within 8 h of collection including Factors V and VIII and this may result in recipient’s RBCs, but
of whole blood is known that are labile, it is useful to control of a bleeding crossmatching is not
as FFP control bleeding in patients episode performed prior to
who have multiple coagulation transfusion; Rh type is
factor deficiencies; FFP should not a consideration
not be used to correct a
deficit of blood volume; other
volume expanders that are less
potentially infectious should
be used
Cryoprecipitate Cryoprecipitate is This component is used for Transfusion of Crossmatch testing

generated by thawing patients with a deficiency cryoprecipitate to is unnecessary;
FFP at 1°C-6°C; the of fibrinogen (often from raise fibrinogen although ABO-
precipitate is collected disseminated intravascular levels to greater than compatible
and refrozen; a typical coagulation), or deficiency of 100 mg/dL may be cryoprecipitate
bag of cryoprecipitate Factor XIII useful to provide is preferred, it is
contains at least 80 units hemostasis for not necessary; the
of Factor VIII and at least fibrinogen deficiency Rh type is not a
150 mg of fibrinogen in a consideration
volume of less than 25 mL
Platelets collected Platelets obtained by Platelet transfusions are The elevation of the Although
by apheresis apheresis contain at least indicated for patients who platelet count in a crossmatching is not
3 × 1011 platelets/unit; are thrombocytopenic thrombocytopenic necessary, platelet
the product has a volume due to decreased platelet patient or the products that are
between 200 and 400 mL production or blood loss and transfusion of ABO-compatible
for those patients who do not functionally active with the recipient are
have an adequate number platelets into a patient preferred to minimize
of functioning platelets; with dysfunctional the infusion of
platelet transfusions are not platelets can result incompatible plasma
usually effective in conditions in the cessation
associated with rapid platelet or prevention
destruction; platelets may of bleeding
be useful in preventing a
bleeding episode if given
as a prophylactic measure
to patients with very low
platelet counts

TABLE 12–1 Blood Component Descriptions and Indications (continued)

Category Description of Product Major Indications Actions Precautions
Platelet concentrates Platelet concentrates are Same as for apheresis platelet Same as for apheresis Same as for apheresis
(whole blood- obtained from a single platelets platelets
derived platelets) unit of whole blood, and
contain at least 5.5 × 1010
platelets; suspended
in 40-60 mL of plasma,
which is stored at
20°C-24°C
Granulocytes Granulocytes are Granulocytes may be The granulocytes Crossmatching must

collected by collected from a single indicated for the patient who may contribute be performed before
apheresis donor by apheresis; the has both neutropenia and a to the eradication transfusion because
product, which contains documented infection that is of infection in a of the large number
other blood cells as well, is not responsive to therapy; this neutropenic recipient of RBCs in the
in a volume of 200-300 mL product should not be used for product; in addition,
prophylaxis against infection; the granulocytes
in general, it has been more are very labile, so
effective in infants than this product should
in adults be transfused
as soon as possible
after collection
TABLE 12–2 Derivatives of Blood Components and Indications for Their Use
Derivative Description and Indication
Factor VIII Prepared for treatment of hemophilia A; Factor VIII can be purified from human plasma and treated to
remove and inactivate infectious agents; recombinant Factor VIII products are available and commonly
used; some plasma-derived preparations contain significant amounts of von Willebrand factor and are
suitable for treatment of this disease as well
Factor IX Prepared using methods similar to those for Factor VIII; it is used for the treatment of patients with
hemophilia B; recombinant factor IX is available and commonly used; concentrates that contain
Factors II, VII, and X along with Factor IX are also available, and are known as prothrombin complex
concentrates; some of these concentrates are potentially thrombogenic and are therefore not the
preferred product for treatment of hemophilia B
Albumin Prepared from pooled donor plasma as a 5% or a 25% solution in a manner that removes infectious
agents; albumin is used as replacement fluid for plasmapheresis, for hypoalbuminemic patients with
acute lung injury, in conjunction with large-volume paracentesis, for diuresis in patients with ascites
unresponsive to diuretics, in selected patients with subarachnoid hemorrhage to prevent vasospasm,
and in selected burn patients; it should not be used routinely for volume expansion when crystalloid
or synthetic colloid volume expanders such as dextran and hydroxyethyl starch are available
Intravenous immunoglobulin (IV Ig) The IgG fraction prepared from pooled donor plasma is processed to minimize IgG dimerization
and remove or inactivate infectious agents; used as antibody replacement therapy in humoral
immunodeficiency states (see Chapter 3) and in the treatment of selected autoimmune disorders such
as idiopathic or immune thrombocytopenic purpura (ITP)
Rh immune globulin The IgG fraction prepared from pooled plasma from donors with high-titer anti-D treated to remove
or inactivate infectious agents; administered by intramuscular injection to Rh-negative women to
prevent their alloimmunization by the Rh-positive RBCs of their offspring; Rh-negative women should
receive this product at 28 weeks of gestation and again within 72 h of the birth of an Rh-positive baby,
or at the time of abortion, miscarriage, vaginal hemorrhage, ectopic pregnancy, abdominal trauma, or
invasive procedure such as amniocentesis or chorionic villus sampling; an intravenous formulation of
this product is used for treatment of ITP
Antithrombin concentrate Prepared for treatment of patients with low amounts of circulating antithrombin who are susceptible
to thrombosis; antithrombin is purified from pooled human plasma and treated to remove or
inactivate infectious agents
Recombinant, activated Factor VII A recombinant version of activated Factor VII prepared for treatment of patients with acquired Factor
VIII and Factor IX inhibitors and for patients with congenital Factor VII deficiency; widespread off-label
use in bleeding patients with complex coagulopathies is being tempered by poor outcomes in
randomized trials; should be used with caution in patients with prothrombotic tendencies
Blood components also may be donated by a procedure known as apheresis, in which

whole blood is removed from the donor, the component of interest (plasma or platelets most
commonly, but RBCs as well) is removed, and the remaining blood elements are returned to
the donor (see Chapter 2 for a diagram of apheresis). This procedure may be done manually,
but is now usually carried out using an automated device. Using an apheresis instrument, whole
blood is drawn from the donor’s vein as an anticoagulant solution (usually citrate) is added, and
pumped into a centrifuge where it is separated into its components. The component of interest
is drawn into a collection bag, and the rest of the blood is returned to the donor via the same or
a different vein. This process may be discontinuous (filling the instrument, separating the com-
ponents, returning the residual blood, and repeating the cycle) or continuous (using separate
lines to draw blood into the instruments and return it to the donor). The entire extracorporeal
circuit is sealed except at the points of contact with the donor’s vein(s). Apheresis is commonly
used to obtain plasma, usually for further processing into derivatives such as albumin, clot-
ting factor concentrates, and immunoglobulins, as well as to obtain platelets. A unit of apher-
esis platelets (commonly called “single donor platelets”) contains more platelets than a unit
derived from a whole blood donation. Transfusion of a unit of apheresis platelets, which must
contain at least 3 × 1011 platelets, usually elevates an adult patient’s platelet count by 30,000 to
50,000 platelets/μL. Since 1 unit of whole blood-derived platelets must only contain 5.5 × 1010
platelets, the usual adult dose is 4 to 10 units or 1 unit/10 kg. Whole blood-derived platelets are
less expensive to prepare than apheresis platelets because they do not require special equipment
for their isolation. However, apheresis platelets can easily be prepared in such a way that they
contain very few residual white blood cells (“process leukoreduced”), which is an advantage for
some patient groups.
Granulocytes also can be harvested by apheresis for transfusion to patients who are neutro-
penic and suffering from severe infection. Instruments designed to collect RBCs by apheresis
(typically 2 units at a time if the donor meets the somewhat more stringent size and hematocrit
requirements) or various combinations of RBCs, platelets, and plasma are also in use. Finally,
apheresis is used to collect peripherally circulating hematopoietic progenitor cells (HPCs) for
autologous and allogeneic HPC transplantation.
Testing of Donated Blood

Donated blood is held in quarantine following collection while a variety of laboratory tests are
performed using blood specimens obtained from the donor. The ABO and Rh types are deter-
mined on an RBC sample obtained at each donation, and the donor serum or plasma is screened
for the presence of unexpected RBC alloantibodies. The concern is that such alloantibodies could
cause destruction of a transfusion recipient’s RBCs if they express the target antigen. Plasma or
platelets from a donor with an alloantibody are not used for transfusion, although RBCs are gen-
erally safe, particularly if they have been saline washed. Records from any previous donations,
including the results of ABO and Rh typing, are also checked, to reduce the opportunity for donor
or unit misidentification.
Transmission of viruses, Infectious Disease Testing

bacteria, and parasites Transmission of viruses, bacteria, and parasites by transfusion of blood components has been
by transfusion of blood well documented. To minimize infectious disease transmission, blood donors are screened for
components has been
evidence of infection and for participation in activities that may have exposed them to infec-
well documented. To
tious agents. In addition, each blood donation is subjected to several tests for infectious agents
minimize infectious
disease transmission,
before it is made available for transfusion. The tests required for each donation are shown in
blood donors are Table 12–3. Platelets, which are stored at 20°C to 24°C, must also be screened for evidence of
screened for evidence bacterial contamination, which is currently responsible for the majority of transfusion-transmit-
of infection and for ted infections. Several commercial systems have been licensed for the testing of leukoreduced
participation in activities apheresis and whole blood-derived platelets. All donors must be screened once for antibody to
that may have exposed Trypanosoma cruzi, the organism that causes Chagas disease, and deferred indefinitely if they are
them to infectious agents. positive. Thereafter, donors do not need to be retested unless they have a possible exposure, that
is, residence in an endemic area of South or Central America.
TABLE 12–3 Infectious Disease Testing of Donated Blood

Required Optional
Serologic test for syphilis Antibody to cytomegalovirus
Antibody to HIV-1 and HIV-2 Alanine aminotransferase activity for liver function
HIV-1 RNA abnormalities resulting from infection
Antibody to hepatitis C virus (HCV)
HCV RNA
Hepatitis B surface antigen
Antibody to hepatitis B core antigen
Antibody to HTLV-I and HTLV-II

West Nile virus RNAa
Antibody to Trypanosoma cruzib
Screen for bacteria (platelets only)

HIV, human immunodeficiency virus; HTLV, human T-cell lymphotropic virus.
a
Determined locally and may vary by season and/or for detection of WNV-infected mosquitoes by public health surveillance.
b
Testing is done once on all donors and is only repeated if there is another exposure, that is, residence in an endemic area of
South or Central America.
COMPATIBILITY TESTING
Pretransfusion Testing to Assess Donor/Recipient Compatibility
for Blood Components Containing RBCs
Prior to transfusion, the compatibility of donor RBCs with the intended transfusion recipient
must be established (see Tables 12–4 and 12–5 for RBC compatibility issues). Part of this process
involves various serologic tests. However, an equally important part of this process is the proper Misidentification of
identification of the patient when the blood bank specimen is obtained, and again when the trans- patients and mislabeling
fusion is initiated. Misidentification of patients and mislabeling of specimens are the most com- of specimens are the
mon serious errors encountered in transfusion. ABO mistransfusion as a result of this kind of most common serious
error is far more frequent than the transmission of HIV and all of the hepatitis viruses, combined. errors encountered
Compatibility testing includes: in transfusion. ABO
mistransfusion as a result
• The identification of patient and proper labeling of the specimen for compatibility testing. of this kind of error is far
The blood bank specimen (tube of blood) must be labeled at the bedside using the patient’s more frequent than the
armband for identification. The label must include 2 patient identifiers (typically name and transmission of HIV and
medical record number) and the date. There must also be some means of identifying the all of the hepatitis viruses,
phlebotomist (commonly, but not necessarily, by signing or initialing the tube or requisition). combined.
TABLE 12–4 RBC Compatibility

ABO Group of Patient Isoagglutinins Present Compatible Donor RBC Units Incompatible Donor RBC Units
A Anti-B A, O B, AB
B Anti-A B, O A, AB
AB Neither A, B, AB, O None
O Anti-A, anti-B, anti-AB O A, B, AB
RBC D Antigen Acceptable Donor Units Unacceptable Donor Units
Rh-positive Rh(D)-positive, Rh(D)-negative None
Rh-negative Rh(D)-negative Rh(D)-positive

TABLE 12–5 Rh Haplotype Nomenclature

D Phenotype
CE Phenotype RhD Positive RhD Negative “d”
Ce CDe = R1 Cde = r′
cE cDE = R2 cdE = r″
ce cDe = R0 cde = r
CE CDE = Rz CdE = ry
• The determination of the ABO and Rh type of the donor. The collecting facility determines
the ABO group (by front- and back-typing as described below) and Rh type of the donated
unit (and checks prior records). The hospital transfusion service must confirm the ABO
group (front type only) and the Rh type of Rh(D)-negative units that have been received
from the collecting facility.
• The determination of the ABO and Rh type of the patient on a current specimen, and a
comparison to previous records, if any. The ABO group (front and back types) and Rh type
are determined on a current specimen. The specimen must be <3 days old for any patient
who has been transfused or pregnant within the last 3 months; however, many transfusion
services require new specimens every 3 days to keep things simple.
• A screen of the recipient’s serum/plasma for unexpected RBC alloantibodies. If unexpected
antibodies (ie, not anti-A or anti-B) are found, as described below, the antigen specificity of
these antibodies must be identified to establish the risk of a hemolytic transfusion reaction
(HTR) and to help identify potentially compatible donor RBCs that lack the target antigen.
A record check for previously identified alloantibodies must also be made.
• The performance of a crossmatch (see Chapter 2 for illustration of the crossmatch). Several
techniques for performing the crossmatch are described below.
• The identification of the patient when the transfusion is initiated. The patient must once
again be properly identified using the armband to be sure that the unit is intended for the
patient. The armband is the only link between the patient, the specimen, and the blood
component.
ABO Grouping
After the identification of the patient and the specimen for compatibility testing, the most impor-
tant step in assuring the safety of an RBC transfusion is the determination of the ABO group of
the donor unit and the intended recipient. The specificity of the A and B blood group antigens lies
in the presence of carbohydrate structures that are borne by membrane-associated glycoproteins
and glycolipids. The A gene encodes a glycosyltransferase that attaches an N-acetylgalactosamine
residue to the core structure (called “H”) while the B gene encodes an enzyme that transfers a
galactose residue. These 2 different residues impart A or B serologic activity, respectively, to the
After the identification glycoprotein or glycolipid core structure. The O gene, which is a phenotypic recessive, does not
of the patient and encode for an active enzyme, so the RBC of people who are group O are coated with an unmodi-
the specimen for fied H structure. Since the A and B genes are codominant, people who inherit 1 copy of each will
compatibility testing, have both enzymes, and thus both A and B antigens will be expressed on their RBCs.
the most important step During the first year of life, individuals begin to make antibodies to whichever A and B
in assuring the safety antigens they lack. Thus, a person with A antigen on his or her RBCs (group A) has naturally
of an RBC transfusion is occurring anti-B antibodies in the plasma (see Table 12–4). It is the presence of these antibodies
the determination of the (called isoagglutinins because of their ability to agglutinate RBCs in vitro) that makes ABO mis-
ABO group of the donor transfusion so hazardous. The isoagglutinins are largely IgM and fix complement readily. Hence,
unit and the intended
they can cause intravascular hemolysis.
recipient.
Because determining the ABO group is so critical, it is required not only to test for A and B
antigens on the RBCs but also to demonstrate the presence of the appropriate anti-A and anti-
B isoagglutinins in the plasma or serum (see Chapter 2 for a diagram of ABO and Rh typing).
The presence of A or B antigens on patient or donor RBCs is detected by combining them with
reagent “anti-A” in 1 test tube and reagent “anti-B” in another test tube, and then assessing RBC
agglutination. Agglutination with anti-A, for example, indicates the presence of A antigen on the
RBCs. This test is called the “front” or “cell” typing. The plasma of a patient or donor is tested for
the presence of anti-A or anti-B antibodies by combining the plasma with reagent RBCs known to
be either group A or group B, and then assessing RBC agglutination. Agglutination of the reagent
B cells indicates the presence of anti-B isoagglutinin in the plasma, which would be the expected
finding in a person who is blood group A. This test is called the “back” or “serum” typing. The
results of the front- and back-typing must be congruent.
Rh Typing
The second most important antigen system with respect to transfusion safety is the Rh system.
Approximately 85% of Caucasians express the D (or Rh) antigen and are called D (or Rh)-positive
(see Table 12–4). Rh-negative individuals, who lack the D antigen, are vulnerable to development
of an alloantibody to the D antigen, the most immunogenic antigen on human RBCs, if they
are exposed to D-positive RBCs by transfusion or, for a woman, by maternal–fetal hemorrhage.
Anti-D is the most common cause of severe hemolytic disease of the newborn, although the fre-
quency of this complication of pregnancy has been considerably decreased since the advent of Rh
immune globulin. This product is an immunoglobulin fraction obtained by pooling the plasma
of people with high-titer anti-D. When given by intramuscular injection to individuals who have
been exposed to D-positive RBCs (eg, women pregnant with a D-positive fetus), it reduces the
chance of sensitization presumably by binding to D-positive fetal cells, leading to their rapid
clearance from the maternal circulation before an immune response can be generated (see the
section “Hemolytic Disease of the Newborn [HDN]” in Chapter 10).
The Rh type is determined by incubating the RBCs with a reagent antibody to the D antigen.
Rh-positive cells expressing the D antigen are agglutinated by the reagent antibody. RBCs that do
not agglutinate in the presence of the Rh antibody are incubated a second time, usually after the
addition of an enhancer of agglutination. RBCs that do not agglutinate after this second step are
considered to be Rh(D)-negative. A small number of people have RBCs that do not agglutinate in
the first step but are agglutinated after the second, enhanced, incubation step. These individuals are
considered to have the weak D (formerly Du) phenotype. Donors, and usually patients as well, who
are weak D are treated as if they are D-positive, since some weak D RBCs can elicit the formation
of anti-D alloantibodies in D-negative individuals, or can be the target for anti-D alloantibodies.
The RHD gene is located on chromosome 1 immediately adjacent to the highly homolgous
RHCE gene, and the 2 are inherited as a haplotype exhibiting linkage dysequilibrium. The RHD
gene encodes for the D protein that expresses D (“Rh”) antigenic activity. The most common
mechanism for the Rh(D)-negative phenotype (especially among people of Caucasian back-
ground) is the complete absence of the RHD gene. This phenotype is often represented as “d,” but
in fact there is no “d” gene or “d” protein. These individuals lack the D protein altogether. The
RHCE encodes for a protein that is structurally very similar to the D protein, but carries 2 differ-
ent antigens, each of which has 2 common, codominant alleles: C/c and E/e. Since these genes are
inherited as a haplotype, a shorthand nomenclature is in wide use and is shown in Table 12–5.
The Antibody Screen and the Indirect Antiglobulin Assay

Used to Detect Antibodies
To determine if the patient has an alloantibody to a RBC antigen, an antibody screen is per-
formed. In this test, the patient’s serum or plasma is combined with 2 or 3 reagent RBCs that are
specifically chosen because they bear a number of the antigens to which clinically significant RBC
alloantibodies are made. These cells are group O so that they will not be agglutinated by the anti-A
or anti-B isoagglutinins that may be present. If the patient serum does not produce agglutination
of the reagent screening cells, then no unexpected RBC alloantibodies are present.
Although the anti-A and anti-B isoagglutinins are predominantly IgM and readily produce
agglutination in vitro, most of the other clinically significant RBC alloantibodies are IgG and
do not. To detect IgG alloantibodies, an assay called the indirect antiglobulin test (formerly the
indirect Coombs test) is used in the antibody screen (see Chapter 2 for a diagram of the indirect
antiglobulin test). In this technique, the patient’s serum is combined with the reagent screening
cells, often in the presence of an additive, such as low ionic strength saline or polyethylene glycol,
which promotes binding of antibody to RBCs, and the mixture is incubated at 37°C. If an RBC
alloantibody is present, it will bind to the screening cell with the target antigen. The cells are then
washed with saline, and the “antiglobulin reagent” is added. Antiglobulin reagent consists of a
mixture of antibodies to IgG and/or complement. These antibodies bind to any IgG or comple-
ment attached to the screening cell. By binding to IgG or complement on adjacent target cells, the
anti-IgG “crosslinks” the RBCs and produces RBC agglutination in vitro. It is called the indirect
antiglobulin test because it requires first an incubation with the alloantibody (the serum sample)
followed by a second step when the antiglobulin reagent is added. The antiglobulin test is com-
monly performed in a test tube, but has also been adapted to assays based on solid phase or gel
column techniques.
If 1 or more of the screening cells is agglutinated by the patient’s serum, indicating the
presence of an RBC alloantibody, steps must be taken to identify its specificity by determining
its target antigen. This is accomplished again using the indirect antiglobulin test and adding
the patient’s serum to a panel of group O RBCs (typically around 10) that have been chosen to
express the target antigens of the most commonly encountered clinically significant alloantibod-
ies. The pattern of which panel cells are agglutinated in the indirect antiglobulin test can be used
to determine the antigen to which the patient’s alloantibody is directed. Based on the accumu-
lated clinical experience with alloantibodies of a given specificity, it is usually possible to predict
the likelihood that a particular alloantibody will cause a hemolytic transfusion reaction (HTR)
or hemolytic disease of the newborn (see Table 12–6). If the alloantibody has the potential of
TABLE 12–6 The Major RBC Antigens: Frequencies and Clinical Significance
Antigen Frequency Antigen Frequency Implicated in Hemolytic Implicated in Hemolytic
System Antigen in Caucasians in Africans Disease of the Newborn Transfusion Reactions
Rh D 0.85 0.92 Yes Yes
C 0.68 0.27 Yes Yes

c 0.80 0.96 Yes Yes
E 0.29 0.22 Yes Yes
e 0.98 0.99 Yes Yes
Kell K 0.09 0.02 Yes Yes
k 0.99 0.98 Yes Yes

a
Duffy Fy 0.66 0.10 Yes Yes
b
Fy 0.83 0.43 Yes Yes
(a–b–)
Fy Rare 0.68 NA NA
a
Kidd Jk 0.77 0.91 Yes, mild case Yes
b
Jk 0.72 0.23 Yes, mild cases Yes
MNSs M 0.78 0.70 Yes, few cases Yes, few cases
N 0.72 0.74 Yes, rarely No
S 0.55 0.31 Yes Yes
s 0.89 0.97 Yes Yes

a*
Lewis Le 0.22 0.23 No Yes, few cases
b*
Le 0.72 0.55 No No
(a–b–)
Le 0.06 0.22 NA NA
NA, not applicable.
*
Not allelic pair.
causing hemolysis, donor RBCs that lack the target antigen must be chosen for transfusion. The
typing of RBCs for specific antigens is accomplished in a manner similar to that used for the
determination of the ABO and Rh type using commercial antisera directed at specific antigens.
Patients who have multiple alloantibodies, or alloantibodies directed at high-frequency anti-
gens, may pose the challenge that very few donors will lack the target antigen(s). Under these
circumstances, the blood bank may have to screen the red cell inventory for antigen-negative There are 3 crossmatch
units, request their blood supplier to do the same, or, in some instances, locate suitable units techniques in common
through a national rare blood registry. use: the antiglobulin
technique crossmatch,
the immediate spin
The RBC Crossmatch crossmatch, and the
There are 3 crossmatch techniques in common use: the antiglobulin technique crossmatch, the electronic crossmatch.
immediate spin crossmatch, and the electronic crossmatch.
The antiglobulin crossmatch was the standard for years and still must be performed when a
patient has an RBC alloantibody or even a history of having had one (see Chapter 2 for a diagram
of the blood crossmatch). This crossmatch procedure is very similar to the antibody screen and
is based on the indirect antiglobulin technique, except in this case the patient’s serum is com-
bined with RBCs from the donor unit. If the patient has an alloantibody to the donor RBCs, the
antibody will become bound to the donor RBCs during the incubation step, and the cells will
be agglutinated by the antiglobulin reagent added in the final step. If agglutination occurs, the
crossmatch is incompatible and the unit of RBCs should not be transfused to that patient. If the
RBCs from this donor were mistakenly transfused, they would be destroyed prematurely, that is,
an HTR would occur. If there is no agglutination, the patient does not have alloantibodies to the
antigens present on this donor’s RBCs, and the crossmatch is compatible.
The immediate spin crossmatch is done by combining the patient’s serum with a sample of
the donor RBCs intended for transfusion, centrifuging them without incubation or the use of the
antiglobulin reagent, and observing them immediately for agglutination. This technique detects
ABO incompatibilities, but is not sensitive to the presence of other RBC alloantibodies. It may
only be used in patients who do not have unexpected alloantibodies (ie, they have a negative
antibody screen), in massive transfusion (transfusion of the equivalent of 1 entire blood volume),
and in emergency circumstances when an abbreviated crossmatch procedure is imperative for
providing blood rapidly.
In the electronic crossmatch, blood bank personnel rely on the computer to verify the ABO
(and Rh) compatibility between donor RBCs and the patient. A number of requirements must be
met by the information system and the bench procedures involved in the typing, and extensive
validation must be performed. This technique is again only suitable for patients who do not have
unexpected RBC alloantibodies, or in emergency situations.
Direct Antiglobulin Test (Formerly Direct Coombs Test)

This test detects the presence of IgG or complement that is bound, in vivo, to the patient’s RBCs,
by using antiglobulin reagent specific for IgG or various complement components including C3b,
C3d, and/or C4d. In this technique, the patient’s RBCs are washed with saline, and the antiglobu-
lin reagent is then added directly (hence the name of the test) (see Chapter 2 for a diagram of the
direct antiglobulin test). The cells are observed for agglutination after incubation. The presence of
RBC coated with immunoglobulin and/or complement is evidence of immune-mediated hemo-
lysis. Disorders associated with a positive direct antiglobulin test include hemolytic disease of the
newborn, autoimmune hemolytic anemia, and drug-induced hemolytic anemia. A positive direct
antiglobulin test result is also observed in patients experiencing an HTR where donor RBCs are
circulating coated with the recipient’s alloantibody. Note that most patients with positive direct
antiglobulin tests do not have hemolysis. A positive direct antiglobulin test is found in many
patients with lymphoproliferative and autoimmune disorders, or who are taking various medica-
tions such as procainamide, vancomycin, and drugs in the penicillin and cephalosporin families.
If a patient has an RBC autoantibody, especially if it is an IgG, it may interfere with routine
serologic testing, especially any test that is based on the indirect antiglobulin technique such as
the antibody screen and the crossmatch. Absorption techniques are used to remove the autoanti-
body from the patient’s plasma, but leave any alloantibody behind. In the autologous absorption
technique, the patient’s RBCs (after treatment to remove any autoantibody) are incubated with
the patient’s plasma. The autoantibodies bind to the patient’s RBC, but any alloantibodies present
do not since the patient lacks those antigens, by definition. After absorbing out all of the autoan-
tibody (which may take a few cycles with fresh batches of the patient’s RBCs) the autoabsorbed
plasma can be tested for alloantibodies using the conventional antibody screen described above.
If the patient has been recently transfused, however, this absorption technique cannot be used,
because the transfused cells might absorb some of the alloantibody as well as the autoantibody
if they happen to bear the target antigen. In this case the RBCs used for the absorption may be
phenotypically matched to the patient, or several cells may be chosen, each of which displays a
different array of RBC antigens—hence the name, the heterologous absorption technique. The
sample of patient plasma that was absorbed by the heterologous cells is then also tested for RBC
alloantibodies using the conventional antibody screen described above.
Compatibility Testing for Other Blood Components

That Do Not Contain RBCs
Compatibility testing for blood components without RBCs (ie, platelets and plasma) is much less
complex than it is for products with RBCs since no crossmatching must be done. ABO grouping
of donor units and the patient must be performed to avoid the transfusion of plasma that is ABO-
incompatible with the recipient’s RBCs. The amount of anti-A and/or anti-B isoagglutinin in a
unit of apheresis platelets or FFP (200-300 mL) could lead to destruction of some of the recipi-
ent’s RBCs if there were an ABO mismatch. Rh-negative recipients may receive plasma products
or apheresis platelets from a donor of any Rh type, since these components do not contain RBCs.
Whole blood-derived platelets from Rh-negative donors may be preferentially selected for Rh-
negative patients, particularly if there is visible RBC contamination of the platelet product, to
avoid the possibility of alloimmunization to the D antigen.
Molecular Techniques in Immunohematology

In the last 20 years, molecular techniques have greatly increased our understanding of blood group
antigen structures and their genetics, and have explained many of the serologic conundrums that
baffled blood bankers for decades. Although not in routine use in the hospital transfusion service
at this point, a widening array of applications has been making its way into the clinical arena.
Some of these applications include:
1. Genotyping blood donors—Microarray-based platforms and mass spectrometry techniques
have been used to genotype large numbers of blood donors for a number of the most
common clinically significant antigens. The availability of this information facilitates the
identification of donor units for patients who require RBC with a specific phenotype.
2. Genotyping patients—Similar technology can be applied to individual patients in
circumstances when it is difficult to obtain a reliable phenotype by serologic methods, for
example, in patients who have already been transfused or who have autoantibodies.
3. Hemolytic disease of the newborn—Detection of the RHD gene in the fetus of an
alloimmunized mother can be performed using amniotic fluid or maternal plasma, thereby
establishing whether or not the fetus is at risk for hemolytic disease of the newborn. The
absence of the RHD gene in the fetus also obviates the need for additional, more invasive
testing of the fetus, such as per-umbilical blood sampling. It is also possible to determine
whether or not the father is homozygous for the RHD gene.
4. Genotyping in the absence of serologic reagents—Typing sera for some clinically
significant blood group systems, such as Scianna and Dombrock, are not routinely
available, and others are periodically in short supply. Genotyping has been used as an
alternative in these situations.
Other potential applications:
1. Extended electronic crossmatching—The use of the electronic crossmatch to insure
ABO and RhD compatibility between donor RBC and recipient is well established.
The extension of this technique for matching for other clinically significant antigens
would be feasible if more extended genotype information was available for donors.
This approach could be used in 2 circumstances:
a. Alloimmunized patients—For example, the database could be searched for donor RBCs
that were A, R1r, Kell (K1) negative for a patient who was group A with anti-E and
anti-K. Only these units would require antigen confirmation (serologically according to
current regulations) and crossmatching.
b. Multiply transfused patients—Prospective genotype matching could be performed
for transfusion-dependent patients, such as those with sickle cell disease, thalassemia,
or aplastic anemia, to reduce the incidence of alloimmunization and delayed HTRs,
especially the hyperhemolysis syndrome.
2. Autoimmune hemolytic anemia—The evaluation of patients with autoimmune hemolytic
anemia is time-consuming and technically demanding, especially if they require autologous
or heterologous absorptions. The goal of identifying units suitable for transfusion could be
met, and perhaps more rapidly, by extended genotype matching.
INDICATIONS FOR TRANSFUSION

Table 12–7 is a list of indications for transfusion of RBCs, platelets, plasma, and cryoprecipitate.
Red Blood Cells Hemoglobin levels

exceeding 10 g/dL
A National Institutes of Health (NIH) Consensus Conference established broad parameters for (100 g/L) rarely require
perioperative RBC transfusion. Although the conclusion of the conference was that “no single transfusion, while those
measure can replace good clinical judgment as the basis for decisions regarding perioperative with hemoglobin levels
transfusion,” it was suggested that patients with hemoglobin levels exceeding 10 g/dL (100 g/L) less than 7 g/dL (70 g/L)
frequently do.
TABLE 12–7 Indications for Transfusion

Packed RBCs
Hgb <7 g/dL or hematocrit <21% in a patient with uncompromised cardiovascular function
Hgb <10 g/dL or hematocrit <30% in a patient with cardiovascular disease, sepsis, or hemoglobinopathy
Platelets
Prophylactically for platelet count <10,000/μL (adults), or <50,000/μL (neonate)
<30,000 platelets/μL and bleeding or minor bedside procedure
<50,000 platelets/μL and intraoperative or postoperative bleeding
<100,000 platelets/μL and bleeding post cardiopulmonary bypass

Do not transfuse platelets in setting of thrombocytopenic thrombotic purpura, heparin-induced
thrombocytopenia. Platelet transfusions are unlikely to be useful in idiopathic thrombocytopenic purpura or
posttransfusion purpura
Fresh frozen plasma
Bleeding in patients with INR ≥2
Bedside procedure and INR ≥2
Prophylaxis (nonbleeding) with INR ≥10
FFP is not indicated for patients with INR <1.5

Thrombotic thrombocytopenic purpura
Cryoprecipitate
Bleeding in the setting of:

• Dysfibrinogenemia
• Fibrinogen <100 mg/dL
• von Willebrand disease
Hgb, hemoglobin; INR, international normalized ratio.
rarely require transfusion, while those with hemoglobin levels less than 7 g/dL (70 g/L) frequently
do. Several professional organizations have also established guidelines for RBC transfusion. There
have been a small number of randomized trials comparing the clinical outcomes of liberal and
stringent RBC transfusion triggers that have consistently failed to demonstrate any benefit of trans-
fusing patients for hematocrits of 30% (10 g/dL) compared with triggers as low as 21% (7 g/dL).
Platelets
Indications for platelet transfusion have also been addressed by an NIH Consensus Conference
and by professional organizations. Of particular interest in the last several years has been a reassess-
ment of the use of prophylactic platelet transfusions in thrombocytopenic patients with marrow
failure. In general, the traditional trigger level of 20,000 platelets/μL for prophylactic transfusion
has been replaced with a level of 10,000 platelets/μL. There has even been a challenge to the utility
of any prophylactic platelet transfusion, including before minor procedures such as line placement
In general, the traditional and lumbar puncture. This challenge suggests that platelets should only be administered in cases of
trigger level of 20,000 actual bleeding. Appropriate uses of platelets in other settings are included in Table 12–7.
platelets/μL for
prophylactic transfusion Fresh Frozen Plasma
has been replaced with a
Clinical situations in which FFP is likely to be useful also have been established by an NIH Con-
level of 10,000 platelets/μL.
sensus Conference and professional organizations. FFP has been used as replacement therapy
for deficiencies of clotting factors and regulatory proteins, including protein C and protein S, for
which specific concentrates or recombinant products are not available. The use of FFP to reverse
mild coagulation abnormalities is probably not warranted. The risk of bleeding appears to be very
low when the prothrombin time (PT) and the international normalized ratio (INR) derived from
it are only mildly elevated (PT is <1.5 times the control or the INR is ≤1.5). The same can be said
for mild elevations of the partial thromboplastin time (PTT) associated with coagulation factor
deficiencies. It is also unlikely to provide any benefit to patients with mild elevations in the PT or
PTT who are undergoing minor procedures (eg, line placement). On the other hand, FFP is effec-
tive in the treatment of thrombotic thrombocytopenic purpura, reversing the effects of warfarin
in an emergency situation, the treatment of the bleeding patient with disseminated intravascular
coagulation, and massive transfusion cases.
Cryoprecipitate
The practice of using cryoprecipitate as a source of fibrinogen and Factor XIII is well accepted. In
addition, cryoprecipitate can be mixed with thrombin to form topical fibrin “glue,” which is used
to initiate anatomic connections and control bleeding over large surfaces; however, products with
standardized amounts of fibrinogen that have undergone viral inactivation procedures are now
commercially available and are generally preferable. The role of cryoprecipitate in the treatment
of bleeding in uremic patients is controversial. Cryoprecipitate is no longer recommended for
treatment of hemophilia A or von Willebrand disease because of the availability of other products.
COMPLICATIONS OF BLOOD TRANSFUSION

An adverse effect of blood transfusion occurs in approximately 3% of transfusions in the United
States. These complications of transfusion can be classified as immunologic, infectious, or due to
the chemical or physical characteristics of blood components.
Immunologic Reactions
RBC Reactions
Hemolytic Transfusion Reactions
Although HTRs are much discussed, they are fortunately quite uncommon, reflecting the efficacy
of the serologic and procedural techniques in place to prevent their occurrence. Although HTRs
occur with less than 0.1% of the units transfused in the United States, they can be life-threatening.
It bears noting that fatal, acute HTR due to ABO incompatibility is a more frequent adverse
outcome of transfusion than infection with HIV or HCV, and it is more often the result of patient An adverse effect of
or sample misidentification than to serologic mishaps or exotic blood types. blood transfusion occurs
HTRs are mediated by antibodies directed against alloantigens present on transfused RBCs. in an estimated 3.0%
Most alloantibodies to RBC antigens, other than the AB isoagglutinins, develop in response to to 3.5% of transfusions
exposure to allogeneic RBCs by transfusion or maternal–fetal hemorrhage. There are hundreds in the United States.
of RBC antigens comprising more than 50 systems. Fortunately, only a small proportion of these These complications
have clinical significance. In addition to the AB isoagglutinins, antibodies to antigens in the Rh, of transfusion can be
Kell, Duffy, Kidd, and MNSs systems are responsible for the preponderance of HTRs. Identifica- classified as immunologic,
infectious, or due to the
tion of these alloantibodies, by the techniques discussed above, is important because the degree
chemical or physical
and severity of hemolysis differs among them.
characteristics of blood
HTRs can be either acute, occurring within 24 hours of transfusion, or delayed, in a reac-
components.
tion that appears 5 to 7 days (range 3-21 days) after the transfusion. Acute reactions are usually
more severe than their delayed counterparts, and occur in patients who already have antibodies
to RBC alloantigens when they are transfused with RBCs bearing those target antigens. The most
severe acute HTRs are due to ABO incompatibility because the AB isoagglutinins are present at
a substantial titer and fix complement efficiently, being largely IgM. The A and/or B antigen sites
are also abundant on RBCs (typically 1-2 × 106 antigen sites per cell). Antibodies to antigens in
the Kell, Kidd, and Duffy systems also have been responsible for acute HTR.
Patients with an acute HTR typically present with temperature elevation, an important point,
because they might initially be mistaken for a febrile-nonhemolytic transfusion reaction (FNHTR;
discussed later). Nausea, vomiting, hypotension, low back pain, and substernal pressure may also
signal the occurrence of acute hemolysis. Hemolysis is generally intravascular in this setting. The
hemoglobin released into the plasma from the lysed RBCs is apparent as hemoglobinemia (red
plasma rather than yellow) and hemoglobinuria (red urine that remains red after centrifugation).
Disseminated intravascular coagulation and systemic hemodynamic instability may be triggered
by the hemolysis. Together with the direct toxic effects of cell-free hemoglobin on the tubular cells
of the kidney, these conditions are responsible for the impaired kidney function that often accom-
panies acute intravascular hemolysis. Therapy is largely supportive, but preservation of renal func-
tion is critical, and is often accomplished through the use of intravenous hydration and diuretics.
Delayed HTRs occur in 2 situations. In one, the patient is exposed to a foreign RBC alloan-
tigen by transfusion and mounts a primary immune response. As the amount of antibody in the
plasma increases, hemolysis may ensue. The second situation in which a delayed response may
occur is when a patient is reexposed to an alloantigen to which he or she was sensitized in the past
by previous transfusion or pregnancy (anamnestic response). Even if the alloantibody to this anti-
gen is not detectable prior to the transfusion, exposure to the alloantigen can stimulate an anam-
nestic response. Antibodies to Kidd and Rh antigens are frequently responsible for such reactions.
Hemolysis is typically extravascular in delayed HTR with the only clinical and laboratory signs
being a decrease in the hemoglobin level, a rise in the bilirubin level, a low-grade temperature,
and a feeling of malaise. When no hemolysis can be detected in a delayed HTR, the reaction is
called a delayed serologic (rather than hemolytic) transfusion reaction.
Reactions to Plasma Components

Hypersensitivity Reactions—Allergic and Anaphylactic Transfusion Reactions
Allergic reactions occur in approximately 1% to 3% of patients receiving blood products con-
taining plasma. In most cases, these hypersensitivity reactions are a host response to foreign
plasma proteins in the donor blood components. The vast majority of these reactions consist of
hives, pruritus, and erythema, and can be managed with antihistamines or steroids. More serious
responses such as bronchospasm, laryngeal edema, gastrointestinal disturbance (nausea, vomit-
ing, cramps, and diarrhea), and hypotension (anaphylactoid reaction) are much less frequent.
IgA-deficient patients with anti-IgA antibodies in their plasma are at risk for serious reactions
including frank anaphylaxis if exposed to IgA in a transfused blood component. If transfusion is
required, these patients should be provided with components from IgA-deficient donors, or, in an
elective situation, store their own components for later use. Washing packed RBC can effectively
remove IgA. Patients who are IgA deficient, but who do not have anti-IgA, do not require special
preparations, but should be observed closely during transfusion.
White Blood Cell Reactions

Febrile-nonhemolytic Febrile-nonhemolytic Transfusion Reactions
transfusion reactions These reactions are among the most common transfusion-related complications and accompany
are among the most approximately 1% to 3% of transfusions of cellular components. They are more common in multi-
common transfusion- ply transfused patients and with nonleukoreduced cellular components. An FNHTR usually pres-
related complications and ents with a temperature elevation of 1°C or more, during or shortly after a transfusion (usually
accompany 1% to 3% of within 1-2 hours), that is unlikely to be associated with the patient’s underlying disease or therapy.
transfusions of cellular The temperature elevation is often accompanied by chills, rigors, and generalized discomfort, and
components. in some patients, nausea and vomiting as well. The majority of these reactions are mild and do not
persist for more than 8 hours. Antipyretics may be administered, and occasionally meperidine
may be required to treat severe rigors. These reactions have long been considered to be the prod-
uct of antileukocyte antibodies present in the recipient’s plasma, reacting with WBCs or WBC
fragments in the transfused product. There may, however, be other etiologies for the FNHTRs,
including the presence of cytokines released by lymphocytes in the donated unit during storage.
Transfusion-associated Graft Versus Host Disease (TA-GVHD)

Immunocompetent T lymphocytes present in cellular blood components may engraft in an
immunoincompetent transfusion recipient, particularly if cellular immunity is compromised. The
engrafted, allogeneic T cells mount an alloimmune response to cells in the skin and gastrointes-
tinal tract, similar to what occurs in hematopoietic stem cell transplant-associated GVHD. How-
ever, in transfusion-associated GVHD, the donor T cells attack the host cells in the bone marrow
as well, making this complication of transfusion lethal in most cases. Fortunately, T lymphocytes
in cellular blood components can be inactivated by exposure to gamma irradiation, which effec-
tively prevents this complication. Patients at risk for this rare complication include those undergo-
ing hematopoietic progenitor cells (HPC) transplantation or who have hematologic malignancies.
Low-birth-weight infants, infants born with hemolytic disease of the newborn, and fetuses receiving
intrauterine transfusions are also at risk. Patients with congenital T-cell immunodeficiencies (eg,
Wiskott–Aldrich and diGeorge syndromes) have also developed this complication. Cellular com-
ponents from blood relative donors are also routinely irradiated to prevent TA-GVHD that may
occur in the circumstance when the donor is homozygous for an HLA haplotype shared with the
transfusion recipient. In this situation, the transfused T cells remain immunologically invisible to
the otherwise immunocompetent host and, rather than being cleared, they engraft and attack the
host because they recognize the mismatched host haplotype antigens as foreign. Most of the reports
of TA-GVHD in other patients, such a those with solid tumors or who were undergoing surgery,
predate the awareness of this 1-way haplotype match, which is the most likely explanation for the
occurrence of this event in these immunocompetent patients.
Transfusion-related Acute Lung Injury (TRALI)

TRALI is characterized by the development of acute respiratory distress, hypoxia, and bilateral
infiltrates on chest x-ray, often accompanied by fever and hypotension, during or within 6 hours
of completion of a transfusion. To meet the current working definition of TRALI, there must be no
preexisting form of acute lung injury or other risk factors such as sepsis, aspiration, or pneumonia.
Most patients recover completely with supportive care, which may include mechanical ventilation,
and the pulmonary infiltrates usually resolve within 2 to 4 days without long-term sequelae. How-
ever, there is a 5% mortality rate. This complication has been attributed to the presence of antileu-
kocyte antibodies in the plasma of donor blood (often from females with a history of pregnancy)
that react with the recipient’s WBCs. This results in the formation of immune complexes that are
trapped in the pulmonary vasculature and lead to alveolar edema. At present, various steps are
being taken to reduce the incidence of TRALI, including making FFP from predominately male
donors or donors with no history of pregnancy or transfusion, or by testing for HLA antibodies.
Platelet Reactions
Posttransfusion Purpura (See the Section “Bleeding Disorders” in Chapter 11)
This rare complication occurs in patients who lack a common platelet antigen (often HPA-
1A) and have developed an alloantibody by exposure through prior transfusion or pregnancy.
When reexposed to HPA-1A by transfusion of a platelet product or an RBC product containing Transfusion-related
contaminating platelets, these patients appear to develop an anamnestic response and become acute lung injury is
severely thrombocytopenic 7 to 10 days later. Paradoxically, the patient’s own platelets, which are characterized by the
HPA-1A negative, are also cleared. Several explanations have been offered including the observa- development of acute
tion that there is an initial IgM response that reacts with GP IIb–IIIa (essentially a platelet auto- respiratory distress,
antibody) but then “matures” with the production of an IgG with anti-HPA-1A specificity. The hypoxia, and bilateral
reaction is self-limiting, but may be complicated by severe hemorrhage. Steroids and intravenous infiltrates on chest x-ray,
immunoglobulin have been used successfully to manage this immunologic reaction. often accompanied by
fever and hypotension,
Refractoriness to Platelet Transfusions during or within 6 hours
Patients may become sensitized to leukocyte and platelet antigens through transfusion or preg- of completion of a
nancy. Transfused platelets may be cleared rapidly when given to a patient who has preformed anti- transfusion.
bodies directed at foreign platelet antigens or HLA Class I molecules, which are also expressed on
the platelet membrane. As a result, it may be extremely difficult to elevate the platelet count in such
patients. A patient is considered to be refractory to platelet transfusion if the increment measured
between 15 and 60 minutes after the platelet transfusion is lower than expected on 2 occasions.
Note that counts done several hours afterward are not useful for determining which patients are
immunologically refractory. The posttransfusion count may be corrected for the number of plate-
lets administered and the patient’s body surface area (the “corrected count increment”) as follows:
Platelet count increment × Body surface area × 1011
Corrected count increment =
Number of platelets transfused
Here the default for number of platelets transfused is: 1 unit whole blood-derived platelets =
5.5 × 1010 platelets; 1 unit apheresis platelets = 3 × 1011 platelets.
A corrected count increment of <7500 is a strong evidence of immunologic refractoriness. Note
that other causes of refractoriness should be ruled out, among them: active bleeding, fever, sepsis,
splenomegaly (splenic sequestration), disseminated intravascular coagulation, marrow transplan-
tation, antibiotics (eg, vancomycin), IV amphotericin B, thrombotic thrombocytopenic purpura,
idiopathic or immune thrombocytopenic purpura, and heparin-induced thrombocytopenia.
Patients with immunologic refractoriness may respond well to platelets from donors who
lack the HLA antigens corresponding to the patient’s HLA alloantibodies (or to platelets that are
HLA matched) or to platelets that have been chosen by platelet crossmatching.
Leukocytes in the transfused unit appear to be necessary for stimulating the immune
response to both platelet and leukocyte antigens. Alloimmunization may be prevented by trans-
fusion of cellular components from which leukocytes have been removed, usually by passage of
the product through a filter that retains the leukocytes. Patients who are likely to need extensive
platelet transfusion support (eg, for HPC transplants or hematologic malignancies) often receive
leukoreduced cellular components to reduce the likelihood of alloimmunization.
Nonimmunologic Reactions
Complications Created by the Physical Characteristics of Blood
Hypothermia
Transfusion of small volumes of cold blood may be associated with minor discomfort. This com-
plication can be averted by using blood warmers or blankets. In the setting of massive transfu-
sion, however, the rapid transfusion of large amounts of blood that is at 1°C to 10°C contributes
to hypothermia. Hemostasis is impaired when the circulating blood is below 37°C and in extreme
situations, cardiac dysrhythmias and arrest may occur. In this setting, the use of high-throughput
blood warmers is warranted.
Transfusion-associated Circulatory Overload (TACO)
Volume overload is a relatively common but often overlooked complication of transfusion.
Patients with congestive heart failure or renal failure, the very young, and the very old are par-
ticularly at risk. It should be suspected in a patient who complains of dyspnea, orthopnea, cough,
or chest pain, during or soon after transfusion, particularly if there are signs of hypoxia, rales,
tachycardia, or hypertension. Supplemental oxygen and diuresis may be required. Future transfu-
sions should be carried out slowly and perhaps with the aid of a diuretic.
Volume overload is Chemical Complications

a relatively common
but often overlooked Iron Overload
complication of Each unit of packed RBC contains approximately 200 mg of iron. Chronic RBC transfusion can
transfusion. Patients with overwhelm the body’s mechanisms for eliminating excess iron, resulting in iron accumulation in
congestive heart failure various tissues. An individual who has received 100 or more units of RBCs (20 g of iron) is at risk
or renal failure, the very to develop various complications of iron overload including cardiac dysrhythmias, pancreatic
young, and the very old failure (“bronze diabetes”), and liver function abnormalities. Tissue iron can be mobilized and
are particularly at risk. excreted using chelating agents such as desferroxamine or deferasirox. Chelation therapy is a slow
process and is more effective if deployed well before tissue accumulation of iron is extensive.
Potassium Toxicity
Potassium leaks out of RBCs during storage as ATP levels decline and the ATPase-dependent
Na+/K+ pump activity diminishes. Once the banked RBCs are transfused, they transport glucose,
restore their ATP levels, and take up the K+ that was lost during storage. In the short term, how-
ever, each unit of RBCs might contain as much as 6 mmol of extracellular K+ at the time of expi-
ration. There have been a handful of reports of neonates, or patients with renal failure receiving
large volumes of banked blood, who have developed life-threatening cardiac dysrhythmias. Neo-
nates usually receive RBC units that have been stored for less than 1 week and have not yet accu-
mulated much extracellular K+. Washing RBC is also an effective means of removing extracellular
K+, although it is very rarely required.
Citrate Toxicity
Citrate is the anticoagulant used in the collection of all blood products and is therefore transfused
with the blood product into the patient. It is present in the plasma. Hence, most of it ends up in
platelet and plasma products while there is relatively little in RBC products. Citrate is metabolized
by every nucleated cell of the body, but in circumstances where large volumes of banked blood
are being infused rapidly, as in massive transfusion, the rapid influx of citrate may overwhelm
the body’s metabolic capacity, leading to an accumulation in the patient’s plasma. Most patients
can receive up to 1 unit of FFP every 6 minutes without evidence of citrate toxicity. Patients
with liver failure metabolize citrate more slowly, however, and are particularly susceptible. The
accumulating citrate chelates calcium, causing the ionized calcium levels to drop and producing
perioral tingling and extremity paresthesias. In extreme circumstances, it may produce severe
hypo(ionized)calcemia that can lead to cardiac dysrhythmias.
Depletion of 2,3-Diphosphoglycerate (2,3-DPG)
With increasing storage time of RBCs, the intracellular level of 2,3-DPG decreases, producing a
left shift of the oxyhemoglobin dissociation curve. Once banked RBCs are transfused, they restore
the levels of 2,3-DPG over a period of 24 to 48 hours. It has been suggested that the high oxygen
affinity of the hemoglobin in the 2,3-DPG-deficient banked RBCs might impair oxygen delivery,
particularly to neonates. As a result, it has become a general practice to transfuse neonates with
RBCs that have been banked less than 1 week. However, most of the literature demonstrating unfa-
vorable outcomes for neonates receiving older units was based on studies with RBC storage sys-
tems in which maintenance of 2,3-DPG levels was not as effective as it is using the current systems.
Infectious Complications (See the Section “Infectious Disease Testing”)

The Classic Pathogens
Transfusion transmission Transfusion transmission of the hepatitis viruses and the retroviruses has been substantially
of the hepatitis viruses reduced through the interventions of donor education, screening on the basis of medical history
and the retroviruses and risk behaviors, and testing, including the use of highly sensitive techniques based on ampli-
has been substantially fication of viral genetic nucleic acids. The residual risk of HIV or HCV infection through trans-
reduced through the fusion is in the range of 1 event per 1 to 2 × 106 units transfused. Viral transmission by pooled
interventions of donor plasma products has also been largely eliminated by the use of robust viral inactivation tech-
education, screening niques or replacement with recombinant proteins.
on the basis of medical
history and risk behaviors, The Current Significant Pathogens
and testing. At the present time, bacterial contamination of blood components is the most significant
infectious complication of transfusion in developed countries, in terms of both the number of
transmitted infections and the number of fatalities. It has been estimated that in the United States,
approximately 1 in 500,000 units of RBCs, or 1 in 10,000 to 20,000 units of platelets, is associ-
ated with transfusion-transmitted sepsis. The organisms most frequently associated with septic
RBC transfusions are psychrophilic gram-negative bacteria such as Yersinia enterocolitica and
Pseudomonas spp., as well as Enterobacter spp. and Serratia spp. Platelet units have been reported
to transmit gram-positive cocci (Streptococcus aureus, S. epidermidis, and Staphylococcus spp.) as
well as gram-negative organisms (Klebsiella spp., Serratia spp., Salmonella spp., and Enterobacter
spp.). The sources of these bacteria are thought to be skin commensals picked up and introduced
into the blood donation with the venipuncture, or less commonly, cryptic bacteremia in clinically
healthy donors. Even if the inoculum is quite small, blood provides a superb culture medium, par-
ticularly when stored at room temperature, as is the case for platelets. Although donors are now
questioned specifically about antibiotic use, the health history is neither a sensitive nor a specific
screening tool. The implementation of tests to screen platelet products for evidence of bacterial
contamination was discussed above and is now routine.
Cytomegalovirus (CMV) is a ubiquitous member of the herpes virus family to which
approximately 30% to 60% of adults in developed countries have been exposed. CMV can be
transmitted by transfusion of blood components that contain leukocytes, such as packed RBCs
and platelets. Although primary infection rarely produces serious disease in immunologically
intact hosts, it is associated with systemic infection in immunocompromised patients who are
CMV-seronegative. The following groups of patients have been shown to be susceptible to trans-
fusion-transmitted CMV primary infection and disease and should receive CMV reduced-risk
cellular blood components:
1. Premature, low-birth-weight (<1200 g) neonates
2. CMV-seronegative pregnant women (including those undergoing intrauterine transfusions)
3. CMV-seronegative recipients of, or candidates for, hematopoietic or solid organ transplants
4. CMV-seronegative, HIV-infected patients
CMV reduced-risk blood components can be obtained by screening donors for CMV anti-
body (IgG) that indicates past exposure, or by removing the leukocytes that contain latent CMV
by filtration with leukocyte reduction filters. These 2 approaches have been shown to be equally
effective in preventing transfusion-transmitted CMV infection. Only cellular components need
to be CMV reduced-risk, since intact mononuclear cells are required to transmit CMV.
Emerging Pathogens
The blood supply will always be vulnerable to the introduction of new pathogens into the donor
population. In some instances, the pathogen may truly be a new organism, or one that has recently
acquired the ability to infect humans, such as the SARS virus, various strains of avian flu, and the
bovine prion responsible for variant Creutzfeldt–Jakob disease. Population shifts in response to
natural or man-made catastrophes, or simply travel for business or pleasure, spread pathogens
from 1 part of the world to another, such as the West Nile virus, Plasmodium spp., and Trypano-
soma spp. In some circumstances, questioning donors about exposure to a pathogen or a history
of a characteristic illness, or the rapid development of a screening test has been an effective means
of interdicting transfusion transmission of a new infectious agent. However, an effective response
is more difficult in the circumstance where the organism has not been identified, its biology is
unique, the routes of transmission are not well understood, or the clinical effects are not well
defined. As a result, work continues to develop pathogen inactivation technology that would be
suitable for cellular blood components.
Transfusion Reaction Workup

If a reaction is suspected, the transfusion must be stopped immediately while maintaining venous
access, and the patient must be assessed. Emergent airway and hemodynamic issues should be
dealt with immediately and appropriate measures taken to alleviate the patient’s major symp-
toms and concerns. If the assessment reveals that the patient’s only symptoms are cutaneous
manifestations of hypersensitivity (flushing, pruritus, and urticaria), then the transfusion may be
resumed under careful observation. In all other situations, the transfusion of that unit should be
stopped and a clerical check should be performed to verify that the correct unit (ie, one labeled
for that patient) has been administered. A transfusion reaction form should be filled out and a
If a reaction is suspected, new blood bank specimen should be drawn from the patient. The transfusion reaction form, the
the transfusion must be unit involved, and the new specimen should be returned to the blood bank for evaluation. A post-
stopped immediately transfusion urine specimen should also be obtained and sent for urinalysis.
while maintaining The blood bank will perform a clerical check, and compare the posttransfusion specimen
intravenous access, and with the pretransfusion specimen used for compatibility testing for the appearance of hemolysis
the patient must be or hyperbilirubinemia. The ABO and Rh type of the posttransfusion specimen will be determined
assessed. to confirm that the pretransfusion specimen was indeed from this patient and that the ABO and
Rh type of the unit that was being transfused was appropriate. A direct antiglobulin test is also
performed on the posttransfusion specimen looking for antibody-coated RBC (ie, donor cells
coated with recipient alloantibody) indicating an immune-based HTR. Any findings suggestive
of an HTR trigger a more extensive investigation in the blood bank. If the workup rules out a
hemolytic reaction, transfusion may resume.
ALTERNATIVES TO ALLOGENEIC TRANSFUSION

The 1980s saw the considerable development of techniques to avoid allogeneic transfusion (trans-
fusion with someone else’s blood), particularly in elective surgery. The major driver was concern
about the infectious complications of transfusion. Before the development of a screening test in
1985, HIV transmission rates may have been as high as 1 in 10,000 units transfused, while as many
as 5% to 10% of transfusion recipients developed what was then called non-A, non-B hepatitis, and
is now known to have been due primarily to HCV, which was only identified in 1989. Although
demand for these blood-sparing techniques is not as great as it was 20 years ago, they are still in
use and continue to be helpful for patients with unusual blood types or multiple alloantibodies for
whom it is difficult to find compatible blood. In addition, the drive to avoid allogeneic blood expo-
sure has reinforced common sense measures: treatment of medically correctable anemia, greater
physician tolerance of asymptomatic anemia, meticulous surgical hemostasis, and the wider use
of hemostatic medications. The licensing of recombinant erythropoietin also reduced the depen-
dence of patients with renal failure, malignancies, and HIV infection on regular RBC transfusion.
Four techniques in particular were developed to reduce the dependence of surgical patients
on banked RBC: preoperative autologous blood donation (PABD), acute normovolemic hemo-
dilution (ANH), intraoperative blood recovery and reinfusion, and postoperative blood recovery
and reinfusion.
PABD is suitable for patients undergoing elective surgical procedures for which RBC transfu-
sion is commonly required, and in this setting can reduce allogeneic blood use. Since the blood
may only be used by the donor/recipient, donor qualifications are simple and no testing (other
than ABO/Rh typing) is required. Note that mistransfusion, bacterial contamination, and volume
overload are just as likely to occur with an autologous unit as with an allogeneic unit. Since the haz-
ards averted (especially infection) are very small, donors who might be put at even a small risk by
donation (eg, mild anemia and coronary artery insufficiency) should be discouraged from PABD.
ANH is a technique whereby several units of blood are removed from a patient in the operating
room immediately before a procedure. The volume is replaced with crystalloid. The blood is returned
if bleeding occurs, or at the end of the procedure. It has the advantage that little advance planning
is necessary, but it is not very efficacious at reducing allogeneic RBC transfusions for patients with
moderate anemia from whom few units can be withdrawn at the beginning of the procedure.
Blood recovered from the operative field can be collected, processed in some manner, and
reinfused. Shed blood is collected by suction into a reservoir, usually with heparin or citrate, and
then usually washed in a centrifugal device specially designed for this purpose. The washed RBCs
are suspended in normal saline and pumped into a bag suitable for reinfusion to the patient. The
washing procedure removes materials that might cause reactions such as cell debris, activated
clotting factors, and complement. A similar process can be carried out manually. This technique
is particularly helpful in procedures where large volumes of blood are lost. Although somewhat
expensive, the recovery of 3 to 4 units of RBCs is usually adequate to recover the costs. This tech-
nique is suitable for elective as well as emergency procedures during which blood loss is extensive.
Devices are also available for collecting blood shed in the postoperative period. Many of
them rely on filtration of the shed blood. The filtration technique is not adequate to remove mate-
rials that can provoke a reaction and is generally not worth risking for the small amounts of blood
that can be recovered. A small, centrifugal device that washes the blood collected postoperatively
is also available. Although it provides a much cleaner product, the small volumes of blood recov-
ered in this manner do not make it very cost-effective.
Cellular therapies
encompass the collection,
CELLULAR THERAPIES processing, storage,
and therapeutic use of
Cellular therapies encompass the collection, processing, storage, and therapeutic use of hemato- hematopoietic cells.
poietic cells, most commonly HPCs. In addition, mononuclear cell fractions from HPC donors
have been used to enhance the graft versus tumor effect of allogeneic transplantation, and dendritic
cells sensitized to tumor antigens have been used to treat solid tumors. Allogeneic HPCs have the
advantage that they are free of malignancy and may have a significant graft versus tumor effect; they
are preferred for most forms of leukemia, Hodgkin disease, and the myelodysplastic syndromes.
Allogeneic transplantation has also occasionally been used to treat certain genetic disorders of the
hematopoietic system, such as sickle cell disease and thalassemia. Autologous HPC transplants are
not complicated by rejection and have a lower incidence of GVHD. They are performed in patients
with some forms of non-Hodgkin lymphoma and multiple myeloma, and as rescue therapy after
intensive chemotherapy for some solid tumors (eg, testicular, breast, and ovarian cancer).
Potential allogeneic donors must in general meet the criteria for blood donation, including
infectious disease testing, although some of these criteria may be waived if an alternate suitable
donor cannot be found. Potential donors must be typed for HLA Class I and II antigens using
molecular techniques. Class I mismatches pose an increased risk for rejection and failure to engraft,
whereas Class II mismatches are associated with increased incidence of GVHD. A single Class I
or II mismatch usually has little impact on survival. Two Class I mismatches, or a Class I and a
Class II mismatch, are usually associated with poorer outcomes. Haploidentical sibling donors
have been used successfully. If a suitable family member donor cannot be found (and only 1 in 4
siblings is likely to be a 2 haplotype match), a donor may be sought through the National Marrow
Donor Program, a registry of people who have been HLA typed and have expressed a willingness
to donate HPCs. The search may take a few months and is less likely to be successful for patients
with unusual phenotypes. There has been considerable effort in the last few years to register donors
from previously underrepresented minority populations. ABO or Rh matching is not necessary
since the transplant recipient will convert to the donor type if engraftment is successful, although
RBC engraftment may be delayed if donor RBCs are incompatible with the recipient’s anti-A or
anti-B isoagglutinins. Donor isoagglutinins may also cause hemolysis of residual recipient RBCs,
or at least a positive direct antiglobulin test. The conversion from recipient to donor blood type
does pose problems for the transfusion service that must provide blood that is compatible with
both donor and recipient until the recipient’s original RBCs and isoagglutinins are undetectable.
HPCs may be collected from peripheral blood by apheresis, from bone marrow by aspiration,
or from cord blood. Apheresis collection now accounts for 90% of autologous transplants, and
50% of allogeneic transplants, a fraction that is increasing. Bone marrow aspiration is performed
with multiple punctures and aspirations of the posterior iliac crest and must be performed in the
operating room with the donor under general anesthesia. The aspirates are anticoagulated (hepa-
rin or citrate), filtered, and pooled into a bag that is then usually stored frozen until the time of
the transplant.
Collection by apheresis is less invasive and less likely to recover residual malignant cells. In
addition, it has been shown to be associated with quicker engraftment, although chronic GVHD
is somewhat more likely to occur than with marrow transplantation. The number of HPCs in the
peripheral blood is ordinarily very low, so donors are prepared by the administration of granulo-
cyte colony-stimulating factor or granulocyte–macrophage colony-stimulating factor at the point
when their marrow is rebounding from a cycle of chemotherapy. Under these circumstances, the
levels of HPCs (which can be determined by measuring the number of CD34-positive cells in the
peripheral blood by flow cytometry) may be elevated 200- to 1000-fold. The pheresis instrument
is configured to collect the mononuclear cell fraction. Large volume collections (with 3 blood
volumes processed) are typically performed, which reduces the number of procedures needed
to collect the targeted number of CD34-positive cells (typically 2-4 × 106 per kg patient weight).
Large volume collection may also have the effect of recruiting HPCs from the marrow during
the collection.
The HPC product undergoes extensive quality control testing including ABO and Rh type,
RBC and WBC counts (and differential), CD34 cell count, an assay to enumerate colony-forming
units in vitro, cell viability, and testing for bacteria, fungi, and mycoplasma. Products are frozen
(usually at a controlled rate) in the presence of 10% dimethylsulfoxide and 10% protein (plasma
or albumin) as cryoprotectants, and stored in mechanical freezers or liquid nitrogen tanks. At the
time of transplant, the units are thawed at 37°C, usually at the patient’s bedside, and then admin-
istered intravenously, much like a conventional transfusion.
Umbilical cord blood contains high levels of circulating HPCs, and this observation has led
to the development of cord blood banking. If a mother meets the criteria for allogeneic blood
donation (except for hemoglobin level and recent pregnancy because she has just delivered)
including the usual infectious disease testing, and there is no history of genetic diseases in the
family of either parent, she may give consent for the blood to be drained from the placenta via the
umbilical cord (after it has been severed or clamped off from the neonate) and then stored frozen.
In addition to the usual quality control testing of the cord blood, HLA Class I and II typing is
performed as well as ABO/Rh typing.
Over 5000 related and unrelated (but HLA matched) cord blood transplants have been per-
formed since the technique was first developed in 1988. Cord blood HPCs home readily to the
host bone marrow and do not seem to be as alloreactive to recipient antigen-presenting cells as
HPCs from adults. In addition, the large numbers of HLA-typed cord blood samples may improve
the chances of finding unrelated matches. Cord transplants are also less likely to be complicated
by GVHD and infection with CMV. However, the total number of HPCs in each cord blood
sample is small and engraftment is slower. This has led to the use of double cord transplants that
accelerate engraftment, even though eventually 1 of the donor’s HPCs dominates.
THERAPEUTIC APHERESIS
Therapeutic apheresis is the process of withdrawing blood from the body, selectively removing
1 particular element (ie, plasma, leukocytes, platelets, or RBCs), and returning the remaining
elements along with a replacement solution (crystalloid and/or colloid) to maintain isovolemia.
There are several different therapeutic apheresis procedures that are designed to remove, or treat,
specific components of the blood. These are described below.
Indications for Therapeutic Apheresis

Indications for therapeutic apheresis have been classified according to the quality of the evidence
demonstrating efficacy or the lack thereof (Table 12–8). The specific disorders for which thera-
peutic apheresis has been evaluated as a treatment are shown in Table 12–9.
TABLE 12–8 Categories of Indications for Therapeutic Apheresis

Category Use of Therapeutic Apheresis Evidence for Clinical Efficacy
Category I Primary therapy or a first-line adjunct Randomized controlled clinical trials or a broad
to other therapies base of published experience
Category II Supportive therapy or a second-line Benefit from apheresis is well accepted, supported
adjunct to other therapies by randomized controlled clinical trials, small series,
or informative case studies
Category III Experimental therapy indicated when Insufficient evidence exists to establish the efficacy
conventional therapies have failed or of apheresis or risk/benefit; trials with conflicting
as part of a research protocol results or small number of case reports
Category IV Lack of therapeutic efficacy of Controlled studies or case reports fail to show
apheresis has been demonstrated clinical benefit
Data from Szczepiorkowski ZM, Winters JL, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice—
evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apheresis.
2010;25:83–177.
TABLE 12–9 Selected Indications for Therapeutic Apheresisa

Disorders Category I Category II Category III Category IV
Solid organ Acute humoral rejection (renal HLA desensitization ABO-incompatible liver
transplantation allograft) (1) (renal allograft) (1) allograft (1)
ABO-incompatible kidney/ Cardiac allograft humoral
heart allograft (1) rejection (1)
Cardiac allograft rejection (2)
Renal Cryoglobulinemia (1) Hemolytic–uremic syndrome Immune complex Diarrhea-associated

Goodpasture syndrome (1) (complement deficiency; rapidly progressive hemolytic–uremic
Rapidly progressive autoantibody type) (1) glomerulonephritis (1) syndrome (1)
glomerulonephritis with Myeloma cast nephropathy (1)
ANCA (1)
Focal segmental
glomerulosclerosis (1)
Neurologic Acute Guillain–Barré Acute CNS multiple Chronic, progressive multiple Amyotrophic lateral
syndrome (1) sclerosis (1) sclerosis (1) sclerosis (1)
CIDP (1) Chronic focal (Rasmussen’s) Paraneoplastic syndromes (1) Inclusion body myositis (1)
Myasthenia gravis (1) encephalitis (1) Stiff person syndrome (1)
PANDAS (1) Lambert–Eaton myasthenic
Paraproteinemic peripheral syndrome (1)
neuropathy (1) Neuromyelitis optica (Devic
Sydenham chorea (1) syndrome) (1)
Acute diffuse
encephalomyelitis (1)
Metabolic Familial hypercholesterolemia Familial hypercholesterolemia Acute heptic failure (1)

(5) (1) Non-mushroom poisoning (1)
Wilson disease—fulminant Mushroom poisoning (1) Refsum disease (1)
with hemolysis (1) Thyrotoxicosis (1)
Sepsis with multiorgan failure (1)
Pancreatitis with
hypertriglyceridemia (1)
Hereditary hemachromatosis (4)
Hematologic Erythrodermic cutaneous ABO-incompatible HPC Nonerythrodermic cutaneous Immune thrombocytopenia

and oncologic lymphoma (2) transplant (1) lymphoma (2) (1)
Hyperviscosity/ Graft versus host disease (2) Posttransfusion purpura (1) Amyloidosis (1)
paraproteinemia (1) Malaria severe (4) Coagulation factor inhibitors (1) Medication-associated
Babesiosis severe (4) Maternal alloimmunization (1) Polycythemia vera (4) thrombotic
Leukocytosis/leukostasis (3) Thrombocytosis (3) Aplastic anemia (1) microangiopathy (1)
Sickle cell crisis (4) Pure red cell aplasia (1) Warm autoimmune hemolytic
Thrombotic thrombocytopenic Cold autoimmune hemolytic anemia (1)
purpura (1) anemia (1) HPC transplant thrombotic
microangiopathy (1)
Autoimmune Catastrophic antiphospholipid Progressive systemic Progressive systemic

antibody syndrome (1) scleroderma (1) scleroderma (2)
Severe systemic lupus (1) Pemphigus vulgaris (2) Pemphigus vulgaris (1)
Dermatomyositis
polymyositis (1, 3)
Psoriasis (1)
Systemic lupus nephritis (1)
ANCA, antineutrophil cytoplasmic antibody; CIDP, chronic inflammatory demyelinating polyneuropathy; CNS, central nervous system; PANDAS, pediatric autoimmune
neuropsychiatric disorders associated with streptococcal infections. Based on information from Szczepiorkowski ZM, Winters JL, Bandarenko N, et al. Guidelines on the use
of therapeutic apheresis in clinical practice—evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apheresis.
2010;25:83–177.
a
Number in parentheses refers to specific apheresis procedure as follows: 1) therapeutic plasma exchange; 2) photopheresis; 3) cytapheresis; 4) red cell exchange; 5) selective
column adsorption.
Therapeutic apheresis has been used as a treatment for numerous disorders. While it is clearly
effective in some diseases, such as thrombotic thrombocytopenic purpura, the therapeutic benefit
of apheresis in many other disorders is much less clear, because many of them are uncommon,
and therefore it is extremely difficult to obtain information about efficacy based on large-scale,
prospective, randomized clinical trials.
Plasmapheresis
In plasmapheresis (plasma exchange; see the figure in Chapter 2), blood is withdrawn from a
patient and the plasma is separated from the cellular components by centrifugation or, less com-
monly, by filtration, in an apheresis instrument. The plasma is discarded and the cellular com-
ponents are returned to the patient. Liters of abnormal plasma can be removed from the patient
and replaced by saline, albumin, starch solutions, FFP, or combinations of these. This technique
is used to remove autoantibodies, immune complexes, paraproteins, and protein-bound toxins.
Cytapheresis
Cytapheresis is the removal of one of the cellular elements of the blood. Leukapheresis is occasion-
ally indicated for patients with acute myelogenous leukemia or chronic myelogenous leukemia
in the accelerated phase with a high level of circulating blasts and evidence of leukostasis with
pulmonary or CNS involvement. Myeloid blast forms adhere to the vascular endothelium and can
impede blood flow in the lungs and the brain. The collection of peripheral HPCs and granulocytes
is a variation of leukapheresis.
Plateletpheresis may be indicated in patients with myeloproliferative disorders, such as essen-
tial thrombocytosis, who develop platelet counts that exceed 1 × 106/μL and also show signs of
hemorrhage or thrombosis.
Erythrocytapheresis (RBC Exchange)

Although most sickle crises are managed with hydration, pain medication, and supplemental oxy-
gen, RBC exchange is occasionally performed for patients who are experiencing a severe infarc-
tive crisis complicated by stroke, acute chest syndrome, retinal infarction, or priapism. Exchange
is performed less commonly to prepare patients for surgery. In the exchange replacement of
sickle RBCs with normal RBCs, the usual goals are to reduce the hemoglobin S concentration
to less than 30% of total hemoglobin, and to increase the hematocrit to 30%. Red cells chosen
for exchange are often screened for hemoglobin S (since donors with sickle trait may be unaware
of it and have a normal hemoglobin level) and may be partially phenotype matched (eg, for Kell
and the Rh antigens) to prevent alloimmunization. Red cell exchange also has been used to treat
patients with malaria or babesiosis who have a high percentage (eg, >10%) of RBCs infected
with organisms despite adequate medical therapy, and signs of decompensation such as marked
hemolysis, pulmonary involvement, CNS involvement, renal failure, or disseminated intravascu-
lar coagulation. Patients who are immunosuppressed, asplenic, or elderly seem to be particularly
at risk to develop complications from infection with Babesia.
Photopheresis
In this apheresis procedure, the patient’s leukocytes are separated from whole blood and exposed
to ultraviolet light, usually after the patient has ingested psoralen. The psoralen/ultraviolet
light-treated leukocytes are then returned to the patient. Photopheresis has been used to treat
cutaneous T-cell lymphoma and has been shown to increase patient survival when compared
with conventional chemotherapy. Photopheresis has also been used to treat GVHD and cardiac
allograft rejection.
A portion of this chapter related to complications of transfusion appeared previously in Clinical

Laboratory Reviews (a newsletter for physicians at the Massachusetts General Hospital, Boston,
1995;4:2).
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C H A P T E R
Diseases of White Blood

Cells, Lymph Nodes,
and Spleen
Daniel E. Sabath
13
LEARNING OBJECTIVES
1. Learn the differential diagnosis of leukopenia.
2. Distinguish between neoplastic and nonneoplastic proliferations of
white blood cells.
3. Learn the diagnostic criteria for the different types of lymphomas,
leukemias, myelodysplastic syndromes, myeloproliferative disorders,
and plasma cell dyscrasias.
4. Understand the genetic, biochemical, and/or cellular defects associated
with the more commonly encountered disorders of WBC function.
CHAPTER OUTLINE
Leukopenia 318 Myeloid Disorders 328
Nonneoplastic Proliferation of WBCs 319 Acute Myeloid Leukemias 328
Lymphocytes 319 Myeloproliferative Neoplasms 333
Eosinophils 319 Chronic Myeloid Leukemia 333
Monocytes 319 Polycythemia Vera 334
Neutrophils 319 Essential Thrombocythemia 335
Neoplastic Proliferation of WBCs 320 Primary Myelofibrosis 335
Lymphoid Malignancies 320 Myelodysplastic Syndromes 335
Lymphoma 322 WHO Classification of the Myelodysplastic
Precursor B- and T-cell Neoplasms 323 Syndrome 336
Chronic Lymphocytic Leukemia 323 Myelodysplastic/Myeloproliferative
Hairy Cell Leukemia 324 Neoplasms 336
Plasma Cell Dyscrasias 324 Disorders Associated With Impaired
Plasma Cell Myeloma 324 WBC Function 337
Waldenström Macroglobulinemia 326 Chediak–Higashi Syndrome 337
Heavy Chain Disease 326 Chronic Granulomatous Disease 337
Monoclonal Gammopathies of Myeloperoxidase Deficiency 337
Unknown Significance 327
Hodgkin Lymphoma 327
A
bnormalities in white blood cells (WBCs) are nearly always quantitative (eg, too many
or too few WBCs). These disorders may be neoplastic, as found in leukemia, or nonneo-
plastic. A qualitative or functional disorder of WBCs may accompany the quantitative
disorder. Qualitative defects in WBC function with a normal WBC count occur, but they are
uncommon. The approach to diagnosis of WBC disorders is shown in Figure 13–1.
317
318 CHAPTER 13 Diseases of White Blood Cells, Lymph Nodes, and Spleen
Determine if the patient has an

WBC count increased and no
infection or other nonneoplastic
evidence of neoplastic disease
disease
History, clinical features, and/or Perform bone marrow biopsy

CBC with blood smear suggestive to evaluate the patient for
of leukemia leukemia
History and clinical features, especially Perform lymph node biopsy of

the presence of lymphadenopathy, affected nodes to evaluate the
suggestive of lymphoma patient for lymphoma
Perform serum protein electrophoresis and,

History, clinical features, and/or
if indicated, immunofixation electrophoresis,
CBC with blood smear suggestive
and bone marrow biopsy to evaluate the
of plasma cell dyscrasia
patient for plasma cell dyscrasia
WBC count is low Determine cause of low WBC count
Patient has history of recurrent Evaluate the patient with battery

infections with normal WBC count of tests for WBC function
FIGURE 13–1 An approach to the patient with a white blood cell disorder.
LEUKOPENIA
A low WBC count can A low WBC count can occur because of a decreased number of lymphocytes, granulocytes,
occur because of a or both. A number of the immunodeficiency diseases are associated with a lymphocytopenia
decreased number (see Chapter 3). Granulocytopenia primarily reflects a reduction in the number of neutrophils
of lymphocytes, (neutropenia) in the peripheral blood. When the number of neutrophils decreases below about
granulocytes, or both. 1000 neutrophils/μL, the neutropenic patient becomes susceptible to infections. These illnesses
range from mild to severe, depending on the type of organism and the effectiveness of the antibi-
otics used to treat it. A classification of granulocytopenic disorders follows.
Defects in the production of granulocytes may be caused by:
• Diseases associated with marrow failure, such as aplastic anemia.
• Diseases in which the marrow is infiltrated by leukemic cells or by metastatic cancer
cells originating from another site; the decreased neutrophil production in this setting is
typically associated with defects in the production of other blood cells as well.
CHAPTER 13 Diseases of White Blood Cells, Lymph Nodes, and Spleen 319
• Suppression of granulocyte production by exposure to certain drugs; the list of drugs that
can produce neutropenia is extensive; noteworthy examples are chemotherapeutic agents
used in cancer treatment and certain nonsteroidal anti-inflammatory drugs (NSAIDs).
• Vitamin B12 or folate deficiency; these disorders produce a megaloblastic anemia and
defective DNA synthesis in granulocyte precursors.
• Suppression of granulocyte production by neoplastic cells, for example, large granular
lymphocytic leukemia.
Accelerated removal of granulocytes may be caused by:
• Immunologically mediated injury to neutrophils following exposure to drugs, with the
injury occurring from an immune response on the neutrophil surface.
• Immunologically mediated injury to neutrophils as part of an autoimmune disorder;
for example, Felty syndrome is a variant of rheumatoid arthritis with neutropenia,
splenomegaly, leg ulcers, and the joint lesions found in rheumatoid arthritis; the
neutropenia can dominate the clinical course in patients with Felty syndrome.
• Immunologically mediated injury to neutrophils that is idiopathic and not associated
with any identifiable abnormality.
• Excessive destruction of granulocytes from splenic sequestration of the neutrophils
in an enlarged spleen or from overwhelming infection.
NONNEOPLASTIC PROLIFERATION OF WBCs

An elevated peripheral WBC count is commonly found in patients with infections and other An elevated peripheral
inflammatory states, such as those associated with autoimmune disorders. WBC count is commonly
found in patients with
infections and other
Lymphocytes inflammatory states, such
Patients can develop a lymphocytosis in a variety of different conditions such as tuberculosis, as those associated with
acute bowel infections, and infectious mononucleosis and other viral infections. autoimmune disorders.
Eosinophils
An increase in circulating eosinophils is most commonly found in patients with allergic disorders and
those with asthma. An increase in circulating eosinophils is also found in patients with certain para-
sitic infections and in patients with dermatologic disorders such as eczema. Increases in eosinophils
can also be caused by some drugs and some autoimmune disorders. Finally, increases in eosinophils
can be seen in certain neoplastic conditions such as Hodgkin lymphoma and T-cell lymphomas.
Monocytes
The peripheral monocyte count is increased in a number of situations where the lymphocyte
count is also increased, such as tuberculosis. Rheumatoid arthritis, systemic lupus erythematosus,
and other connective tissue diseases also may be associated with a monocytosis.
Neutrophils
A mild increase in circulating neutrophils can occur without disease after strenuous exercise,
during menstruation, and in the course of pregnancy. An increased neutrophil count is clinically
significant when it is indicative of a bacterial infection, a neoplastic disorder, ischemia, an auto-
immune disorder, or an effect of certain drugs, such as corticosteroids or epinephrine. The most
frequently identified immature neutrophil in the blood when there is an increased WBC count
is the neutrophilic band cell. The percentage of WBCs represented by band cells or more imma-
ture neutrophil precursors is a commonly used indicator of infection. However, band counts are
poorly reproducible among medical technologists, so the current trend is to not report band
counts. Other less mature neutrophil precursors can be seen in infections and other conditions
where the bone marrow is attempting to produce granulocytes rapidly.
NEOPLASTIC PROLIFERATION OF WBCs

White cell neoplasms WBC neoplasms frequently involve the peripheral blood, and can result in leukocytosis. White
are broadly divided cell neoplasms are broadly divided into 2 large categories, lymphoid (the lymphocyte lineage) and
into 2 large categories, myeloid (the lineage including granulocytes, monocytes, megakaryocytes, and erythroid cells).
lymphoid (the Lymphoid disorders include acute precursor lymphoblastic leukemias (ALL) and mature B-, T-,
lymphocyte lineage) and NK-cell neoplasms. Myeloid disorders include acute myeloid leukemias, myeloproliferative
and myeloid (the neoplasms, and myelodysplastic syndromes.
lineage including
granulocytes, monocytes,
megakaryocytes, and LYMPHOID MALIGNANCIES
erythroid cells).
The lymphoid malignancies are caused by neoplastic transformation of lymphocytes or their
precursors. Lymphoid cells can be found in the lymph nodes, blood, bone marrow, spleen, and
extranodal sites such as the skin, mucosae, and respiratory and gastrointestinal tracts. Lymphoid
neoplasms can occur at any of these sites. Neoplasms that primarily involve the bone marrow
and peripheral blood are referred to as leukemias, and those involving tissue sites are called lym-
phomas. However, many lymphoid malignancies can involve both tissues and the blood/bone
marrow, so the leukemia/lymphoma distinction is somewhat arbitrary. The World Health Orga-
nization (WHO) classification system for lymphoid malignancies is shown in Table 13–1.
Lymphoid leukemias can correspond to precursor B or T cells or mature lymphoid cells.
Precursor lymphoid malignances are also called lymphoblastic leukemias/lymphomas. Relatively
common B- and T-cell malignancies that frequently present in a leukemic phase include chronic
lymphocytic leukemia (CLL)/small lymphocytic lymphoma, hairy cell leukemia, mantle cell lym-
phoma, Burkitt lymphoma/leukemia, T-cell prolymphocytic leukemia, T-cell large granular lym-
phocytic leukemia, and Sézary syndrome. Lymphoid leukemias generally present with elevated
white counts (specifically lymphocytosis), and depending on the degree of bone marrow involve-
ment, there can be decreased numbers of normal white cells, red cells, and/or platelets. Processes
that involve the marrow extensively can result in the presence of myeloid and erythroid precursor
cells in the peripheral blood. Leukemias are diagnosed by examination of the peripheral blood
smear and bone marrow aspirates and biopsies. Additional studies such as immunophenotyp-
ing by flow cytometry, molecular diagnostic techniques, and cytogenetics are frequently used to
establish a diagnosis.
When lymphoid malignancies are mostly tissue-based, they are referred to as lymphomas.
Lymphomas are monoclonal, neoplastic proliferations of B, T, or NK cells. Most lymphomas are
malignancies of mature lymphocytes, but precursor lymphocytic malignances can involve tissues,
and thus be classified as lymphomas. Lymphomas are divided into 2 major groups, Hodgkin lym-
phoma and a much larger variety of lymphomas known generically as non-Hodgkin lymphomas.
The patient with lymphoma often presents with an isolated, enlarged superficial lymph node,
which may be discovered accidentally on physical exam. Alternatively, the patient may have gen-
eralized lymphadenopathy. If the enlarged lymph node develops in a site where it can produce
signs and symptoms, it is more likely to be discovered early in the course of disease. An example
is the enlargement of lymph nodes in the mediastinum, which can impair blood flow through
the large vessels in the chest and produce symptoms on that basis. In some cases, organ involve-
ment may be the first manifestation of a lymphoma. Non-Hodgkin lymphomas, for example, may
become symptomatic when there is cellular proliferation in the orbit, the gastrointestinal tract, or
the skin. Involvement of the bone marrow and peripheral blood also may be an initial indicator
of the presence of a lymphoma.
Lymph node biopsy is the preferred method for diagnosis of lymphoma, since it allows the
pathologist to determine the overall tissue architecture and get a large sample of the cells present.
Because the lymphoma may not be distributed evenly in all lymph nodes, it may be necessary to
biopsy several lymph nodes to establish the diagnosis. In recent years, both fine needle aspira-
tion and biopsy have been used more commonly to make diagnoses of lymphoma. Although fine
needle aspiration does not allow optimal evaluation of tissue architecture, diagnostic procedures
such as flow cytometry and/or molecular techniques can be used to render a diagnosis on mini-
mal amounts of material.
TABLE 13–1 2008 World Health Organization Classification of Lymphoid

Neoplasms
B-cell neoplasms
Precursor B-cell neoplasms

B-cell lymphoblastic leukemia/lymphoma, unspecified
B lymphoblastic leukemia/lymphoma with specific cytogenetic abnormalities
Mature B-cell neoplasms

Chronic lymphocytic leukemia/small lymphocytic lymphoma
B-cell prolymphocytic leukemia
Splenic marginal zone lymphoma
Hairy cell leukemia
Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia
Heavy chain diseases (alpha, gamma, mu)
Plasma cell myeloma
Plasmacytoma (of bone or extraosseous)
Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT)
Nodal marginal zone B-cell lymphoma
Follicular lymphoma
Mantle cell lymphoma
Diffuse large B-cell lymphoma
Mediastinal (thymic) large B-cell lymphoma
Intravascular large B-cell lymphoma
ALK-positive large B-cell lymphoma
Plasmablastic lymphoma
Primary effusion lymphoma
Burkitt lymphoma/leukemia
T- and NK-cell neoplasms
Precursor T-cell neoplasms

Precursor T lymphoblastic leukemia/lymphoma
Blastic NK-cell lymphoma
Mature T- and NK-cell neoplasms
T-cell prolymphocytic leukemia
T-cell large granular lymphocytic leukemia
Aggressive NK-cell leukemia
Systemic EBV-positive T-cell lymphoproliferative disease of childhood
Hydroa vacciniforme-like lymphoma
Adult T-cell leukemia/lymphoma
Extranodal NK/T-cell lymphoma, nasal type
Enteropathy-associated T-cell lymphoma
Hepatosplenic T-cell lymphoma
Subcutaneous panniculitis-like T-cell lymphoma
Mycosis fungoides
Sézary syndrome
Primary cutaneous anaplastic large cell lymphoma
Primary cutaneous gamma-delta T-cell lymphoma
Peripheral T-cell lymphoma, unspecified
Angioimmunoblastic T-cell lymphoma
Anaplastic large cell lymphoma
Hodgkin lymphoma
Nodular lymphocyte-predominant Hodgkin lymphoma

Classical Hodgkin lymphoma
Nodular sclerosis
Lymphocyte-rich
Mixed cellularity
Lymphocyte-depleted
Data from Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.
Lyon, France: IARC Press; 2008.
Principal differentiating factors between Hodgkin and non-Hodgkin lymphomas are:

• Hodgkin lymphoma:
(a) Proliferation of cells is typically localized to a single group of nodes such as the cervical
or mediastinal nodes.
(b) Proliferating cells spread by contiguity.
(c) Mesenteric lymph nodes are rarely involved.
• Non-Hodgkin lymphomas:
(a) Frequent involvement of multiple groups of nodes.
(b) Proliferating cells spread widely and noncontiguously.
(c) Mesenteric lymph nodes are commonly involved.
The Hodgkin and non-Hodgkin lymphomas are classified into clinical stages based on the
distribution of the disease. These stages, with increased clinical severity associated with higher
stage numbers, are as follows:
• Stage I—involvement of 1 group of lymph nodes or 2 contiguous lymph node clusters on
the same side of the diaphragm.
• Stage II—involvement of 2 or more noncontiguous lymph node groups on the same side of
the diaphragm.
• Stage III—lymph node involvement above and below the diaphragm; if there is involvement
of the spleen, the lymphoma is classified as III(s); and if there is visceral involvement by
direct extension, it is known as stage III(e).
• Stage IV—widespread disease, often involving the liver, bone marrow, lungs, bones, and skin.
In addition to the above staging scheme, the designation “B” is added for patients who have
constitutional symptoms such as fever, night sweats, and weight loss. For example, a patient with
involvement of 2 groups of lymph nodes on the same side of the diaphragm with fevers and night
sweats would be considered stage IIB. In general, the presence of these “B symptoms” portends a
more advanced stage of disease with worse prognosis. In addition, the designation “E” is used to
designate lymphomas involving extranodal sites only (eg, the gastrointestinal tract).
Lymphoma
Historically the diagnosis of Hodgkin and non-Hodgkin lymphoma was primarily based on
For Hodgkin lymphoma, the histological appearance of the lymph nodes. For Hodgkin lymphoma, the Rye classifica-
the Rye classification tion system was used for decades and has now been incorporated with relatively few changes
system was used for into the current WHO classification system for hematologic malignancies. Classification of non-
decades and has now Hodgkin lymphomas has been more problematic. Non-Hodgkin lymphomas were organized in
been incorporated with the Rappaport classification in 1966, the Lukes–Collins classification in 1973-1974, and in 1982
relatively few changes they were reclassified according to the Working Formulation of Clinical Usage by an interna-
into the current WHO tional panel of experts.
classification system By the early 1990s, significant progress was made in understanding the biology of lympho-
for hematologic
mas, so newer classification systems were developed based on typing lymphomas with antibodies
malignancies.
specific for cytoplasmic and cell surface proteins (immunohistochemistry and flow cytometry)
Classification of
and by detecting specific molecular lesions. In 1994, the REAL classification was introduced by
non-Hodgkin lymphomas
has been more
the International Lymphoma Study Group. The goal of the new classification was to integrate
problematic. morphological, immunologic, and genetic information to better define the disease entities. The
REAL classification system was modified somewhat to form the basis for the current (2008) WHO
classification system (Table 13–1).
The WHO classification system, like the REAL classification system preceding it, attempts to
classify non-Hodgkin lymphomas according to the normal cell equivalent of the neoplastic cells.
First, neoplastic cells are classified based on whether they are of B-cell or T-cell/NK-cell origin.
Next, the cells are classified by the stage of differentiation to which they correspond. Most B- and
T-cell neoplasms correspond to mature B and T cells.
In the end, lymphoma classification is determined by the architectural features observed
under the microscope (eg, follicular vs diffuse growth pattern and the microscopic appearance of
the malignant cells), the spectrum of proteins expressed on the surfaces and in the cytoplasm of
the malignant cells (eg, T- or B-cell markers and proteins not expressed in normal lymphocytes),
the presence of clonal rearrangements of the immunoglobulin or T-cell receptor genes, and, in
some cases, the presence of specific genetic lesions in the malignant cells. The techniques used
for lymphoma diagnosis include light microscopy, immunohistochemistry, flow cytometry, and
molecular techniques including cytogenetics, fluorescence in situ hybridization (FISH), poly-
merase chain reaction (PCR), and newer techniques such as microarrays.
Since it is beyond the scope of this chapter to discuss all the lymphoid malignancies in detail,
the more common disorders have been selected for inclusion.
Precursor B- and T-cell Neoplasms

Neoplasms of immature B and T cells most commonly present as leukemias, with extensive blood
and bone marrow involvement, but they can also involve the lymphoid tissues as lymphomas. For
example, precursor T-cell leukemia/lymphoma often presents with a mediastinal mass and may
not demonstrate blood or bone marrow involvement. ALL accounts for almost one third of all ALL accounts for almost
childhood cancers and represents 75% of all pediatric leukemias. The median age at diagnosis is one third of all childhood
10 years with a slight male predominance of 1.4:1. Pediatric leukemias are almost always (80%- cancers and represents
85%) of a precursor B-cell lineage, with the remainder being T-cell lineage. 75% of all pediatric
leukemias.
Diagnosis
• Morphology—Morphologically, the involved tissues show monomorphic collections
of medium-sized cells with fine chromatin, high nuclear:cytoplasmic ratios, and
inconspicuous nucleoli.
• Immunophenotyping—Depending on lineage, the cells will express B- or T-cell surface
proteins. Both B- and T-cell precursor cells contain the enzyme terminal deoxynucleotidyl
transferase.
• Cytogenetics—There are a number of recurrent chromosomal translocations associated with
ALL. Hyperdiploidy with 50 or more chromosomes is a favorable prognostic finding. The
presence of the Philadelphia chromosome, t(9;22)(q43;q11), is an adverse prognostic finding.
Chronic Lymphocytic Leukemia

Description
CLL is the most common of the non-Hodgkin lymphomas. The median age at diagnosis is 70 years CLL is the most common
with a slight male predominance of 1.7:1. The neoplastic cells in CLL are mature B cells. CLL is an of the non-Hodgkin
indolent disease with a highly variable life expectancy. Transformation to aggressive disease occurs lymphomas. CLL is an
in 5% to 10% of cases at any time during the course of the illness, and is usually a terminal event. indolent disease with
a highly variable life
expectancy.
Diagnosis
• Morphology—The lymphocytes in CLL are usually small and well differentiated. They are
sometimes difficult to distinguish from normal lymphocytes, but they can be identified
by their somewhat larger size, coarsely clumped chromatin, and tendency to break apart
on peripheral blood smears, forming “smudge cells.” CLL can transform into a high-grade
B-cell lymphoma known as Richter syndrome in approximately 3% of B-cell CLL cases.
Another type of transformation is to the prolymphocytoid form, where patients can have a
very high white count of characteristic prolymphocytes with prominent nucleoli.
• Immunophenotyping—The CLL tumor cells express low levels of monoclonal surface IgM and
IgD in the majority of cases, surface IgM only in approximately 25% of the cases, and surface
IgD, other immunoglobulin isotypes, or no surface immunoglobulin in a small percentage of
cases. A characteristic finding in CLL is expression of CD5, which is normally a pan-T-cell
antigen, but is expressed on a minor normal subset of B cells. CLL cells also express the B-cell
antigens CD19, CD20 (low level), and CD23. Immunophenotyping can also be used for
prognosis: high-level expression of CD38 and ZAP-70 is associated with worse prognosis.
• Cytogenetics—Chromosomal abnormalities in CLL have prognostic significance. Deletions
of 11q and 17p are associated with significantly shorter survival. Deletion of 13q is
associated with better prognosis.
• Molecular genetics—As with all B-cell lymphomas, the cells of CLL have clonally
rearranged immunoglobulin genes. CLL with hypermutated immunoglobulin gene regions
(compared with the baseline unmutated sequences) has a better prognosis. Unmutated
immunoglobulin genes are associated with worse prognosis.
Hairy Cell Leukemia

Description
Hairy cell leukemia is an uncommon form of non-Hodgkin lymphoma. This disease generally
occurs in men with a median age at diagnosis of 50 years. The male to female ratio is approxi-
mately 4:1. The clinical manifestations are primarily the result of infiltration of the tumor cells
into the bone marrow, liver, and spleen. A significant clinical finding on physical examination is
the often massive splenomegaly. The liver is also enlarged, but to a much lesser degree than the
spleen. Marrow failure is common in this disease, resulting in pancytopenia and its associated
complications. Patients generally present with splenomegaly, leukopenia with a relative decrease
in monocytes, and an inaspirable bone marrow.
Diagnosis
The diagnosis of hairy cell • Morphology—The diagnosis of hairy cell leukemia is supported by the identification of
leukemia is supported lymphocytes with bean-shaped nuclei and fairly abundant gray cytoplasm, giving the cells
by the identification of a somewhat monocytic appearance. Fine cytoplasmic projections that have a hair-like
lymphocytes with bean- appearance on Wright–Giemsa-stained smears give this entity its name.
shaped nuclei and fairly • Cytochemistry—The cells in hairy cell leukemia stain positively for acid phosphatase that
abundant gray cytoplasm, is partially or completely resistant to removal on the addition of tartrate. This is known
giving the cells a as TRAP, for tartrate-resistant acid phosphatase. TRAP-positive lymphocytes with fine
somewhat monocytic cytoplasmic projections are highly consistent with a diagnosis of hairy cell leukemia.
appearance. Fine
• Immunophenotyping—The hairy cells have a B-cell phenotype, with monoclonal surface
cytoplasmic projections
immunoglobulin, CD19 (increased), and CD20 (increased). Antigens that are relatively
that have a hair-like
specific for hairy cell leukemia include the interleukin 2 receptor alpha, CD25, as well as
appearance on Wright–
Giemsa-stained smears surface CD11c and CD103. Immunohistochemistry performed on bone marrow biopsies
give this entity its name. or spleen can be used to detect DBA44, which is relatively selective, although not specific,
for hairy cell leukemia. These results are all consistent with the identification of hairy cell
leukemia as a B-cell disorder.
• Molecular genetics—The neoplastic B cells of hairy cell leukemia have clonally rearranged
immunoglobulin genes. Recently most hairy cell leukemias were found to have a mutation
of the BRAF gene (V600E) that was previously found in melanoma. This mutation is
relatively specific for hairy cell leukemia and does not appear to affect disease prognosis.
Plasma Cell Dyscrasias

The plasma cell dyscrasias are disorders in which there is an expansion of a single clone of immu-
noglobulin-secreting cells. This results in the appearance of high levels of complete or incom-
plete immunoglobulin molecules in the serum or urine. The monoclonal immunoglobulin in the
serum is known as an M-component because it is found in the prototype disorder in this group
of diseases, multiple myeloma. Incomplete immunoglobulins containing only light chains or only
heavy chains may be produced in certain plasma cell dyscrasias. The free light chains, which are
known as Bence-Jones proteins, may be excreted into the urine. The 5 disorders included in this
grouping of plasma cell dyscrasias are plasma cell myeloma, Waldenström macroglobulinemia,
heavy chain disease, primary amyloidosis, and monoclonal gammopathy of unknown significance
(MGUS). Amyloidosis is discussed in Chapter 3, and the other entities are described as follows.
Plasma Cell Myeloma

Description
Plasma cell myeloma, also known as multiple myeloma, is a disorder resulting from prolifer-
ation of a single plasma cell clone that produces a monoclonal immunoglobulin. The median
TABLE 13–2 Diagnostic Criteria for Plasma Cell Myeloma

Plasma cell myeloma is divided into symptomatic and asymptomatic forms, depending on evidence of
end-organ damage
Symptomatic plasma cell myeloma:

• M-protein in serum >30 g/L or urine
• Plasmacytoma or clonal plasma cells in bone marrow
• Evidence of end-organ damage including hypercalcemia, renal insufficiency, anemia, or bone lesions
Asymptomatic (also known as smoldering) myeloma:

No evidence of end-organ damage or myeloma symptoms with:
• M-protein in serum >30 g/L
• 10% or more clonal plasma cells in the bone marrow
age for presentation is 62 years. The most frequent presenting symptom is bone pain resulting
from osteolytic lesions produced by clusters of plasma cells infiltrating the bone. The bones most
often affected are the skull, the ribs, the vertebrae, and the long bones of the extremities. Because
patients with multiple myeloma are often anemic, fatigue and weakness are common presenting
symptoms. Patients may also experience recurrent bacterial infections as a result of the leukope-
nia that occurs later in the disease. In addition, the passage of free light chains into the urine may
result in “myeloma kidney” and lead to renal failure. The diagnosis of myeloma depends on the The diagnosis of myeloma
presence of a monoclonal protein in the serum or urine, and then the type of myeloma is further depends on the presence
classified based on the severity of the disease (Table 13–2). A skeletal survey is included in the of a monoclonal protein
initial workup of myeloma to assess the extent of bone involvement. in the serum or urine, and
then the type of myeloma
is further classified based
Diagnosis
on the severity of the
• Morphology—The diagnosis of plasma cell neoplasm is made when increased numbers of disease.
plasma cells are observed in a bone marrow or tissue biopsy. In the bone marrow, plasma
cell numbers will be increased, and they form small clusters to extensive sheets in bone
marrow biopsies. Solitary tissue lesions, often involving bone, may also show sheets of
abnormal plasma cells and are classified as plasmacytomas. Abnormal plasma cells are
rarely seen in the peripheral blood, and “plasma cell leukemia” is considered an end-stage
presentation of this disorder.
• Immunophenotyping—Abnormal plasma cells can be detected by flow cytometry based
on abnormal loss of CD19 and CD45, expression of CD38 and CD138, and monoclonal
immunoglobulin light chain in the cytoplasm. The abnormal cells may express CD56, which
is absent on normal plasma cells. In tissue sections, the abnormal plasma cells are recognized
by expression of CD138 and monoclonal cytoplasmic immunoglobulin light chain
expression.
• Cytogenetics—Chromosomal abnormalities in myeloma have prognostic significance.
Most commonly FISH is used to identify specific abnormalities. For FISH, fluorescent DNA
probes for the genes of interest are used to localize these genes in chromosome preparations
or in cell nuclei. These probes can determine if 2 separate genes are brought together in a
translocation or if a gene is broken apart by a translocation. It is helpful to use some kind
of enrichment technique, such as magnetic beads coated with antibodies that plasma cells
express (eg, CD138), to obtain enough plasma cells to study. Favorable risk cytogenetic
abnormalities include hyperdiploidy, t(11;14) or t(6;14). Poor risk cytogenetic abnormalities
include deletion of chromosome 13, t(4;14), t(14;16), t(14;20), deletion of 17p13, and
hypodiploidy.
• Molecular genetics—Plasma cell neoplasms have clonal rearrangements of their
immunoglobulin genes.
• Protein electrophoresis—The evaluation of a patient for multiple myeloma begins with
protein electrophoresis of serum and urine to identify any monoclonal proteins (see
Chapter 2 for protein electrophoresis and immunofixation). An M-component on an
electrophoretic gel is a dense band of protein that is not usually present. It most often
migrates in the gamma region of the gel, but occasionally appears in the beta or alpha-2
region. To increase the likelihood of M-component detection in the urine, the samples
evaluated for M-components must be concentrated prior to electrophoresis. Confirmation
that a band identified on serum or urine protein electrophoresis represents an
M-component involves further analysis by immunofixation electrophoresis (see Chapter 2)
or, much less frequently now, by immunoelectrophoresis. Both of these tests permit
identification of the specific heavy chain and light chain of the M-component, if both
are present. It is also necessary to quantify serum immunoglobulins to determine if the
concentration of the M-component is greater than 35 g/L.
• Other chemistry tests—Beta-2 microglobulin is the light chain of a class 1 major
histocompatibility complex protein, and is present on the surface of all nucleated cells.
Increased levels of the unbound beta-2 microglobulin in the plasma are found in multiple
myeloma and are considered a reflection of tumor burden. Other tests used to evaluate
myeloma include measurement of serum calcium and evaluation of renal function.
Waldenström Macroglobulinemia
Description
Waldenström macroglobulinemia is the clinical syndrome associated with lymphoplasmacytic
lymphoma in the WHO classification. There is a diffuse infiltration of the bone marrow by small
lymphocytes and plasma cells that synthesize an IgM immunoglobulin, which is referred to as a
macroglobulin. It is similar to plasma cell myeloma in that both have an M-component. However,
The M-component the M-component in Waldenström macroglobulinemia is always an IgM molecule, and unlike
in Waldenström the relatively rare IgM myeloma patient, individuals with Waldenström macroglobulinemia do
macroglobulinemia is not have lytic bone lesions. The mean age for presentation is 63 years, with a slight male pre-
always an IgM molecule, dominance. Patients frequently present with fatigue, weight loss, weakness, and bleeding from
and unlike the relatively anemia and thrombocytopenia. When present in sufficient concentration, the large circulating
rare IgM myeloma IgM protein produces a hyperviscosity syndrome in the plasma and tissue deposition of IgM.
patient, individuals Most patients with Waldenström macroglobulinemia have an elevated serum viscosity, but only
with Waldenström 15% to 20% are symptomatic. The most common symptoms associated with slow blood flow from
macroglobulinemia
hyperviscosity are blurred vision, mucosal bleeding, dizziness, and, on funduscopic examination
do not have lytic bone
of the eye, papilledema, hemorrhage, and distention of the retinal veins.
lesions.
Diagnosis
• Morphology—The pathological correlate of Waldenström macroglobulinemia is
lymphoplasmacytic lymphoma. The abnormal cells are small mature-appearing
lymphocytes, some of which may resemble small plasma cells. The cells can be present in
tissue biopsies, peripheral blood, or bone marrow.
• Immunophenotyping—The abnormal cells express the B cell markers CD19 and CD20
without coexpression of CD5 or CD10. The cells will have surface expression of monoclonal
immunoglobulin. The abnormal cells may express plasma cell antigens such as CD38 or CD138.
• Protein electrophoresis—The diagnosis of Waldenström macroglobulinemia requires
demonstration of an IgM serum protein concentration greater than 30 g/L. As with multiple
myeloma, Waldenström macroglobulinemia must be differentiated from an IgM MGUS.
• Molecular genetics—Recently a mutation in the MYD88 gene (L265P) has been found to
be specifically associated with Waldenström macroglobulinemia.
Heavy Chain Disease

Description
The heavy chain diseases are a group of lymphoproliferative disorders in which there is produc-
tion of monoclonal immunoglobulins with only heavy chains. Each type of heavy chain disease is
named for the abnormal heavy chain produced, resulting in:
• Alpha chain disease—a high serum concentration of the heavy chain present in IgA
• Gamma chain disease—a high serum concentration of the heavy chain present in IgG
• Mu chain disease—a high serum concentration of the heavy chain present in IgM
All the heavy chain diseases are rare, with alpha chain disease having the highest inci-
dence of the related disorders. In all 3 disorders, the monoclonal heavy chain is defective with
internal deletions of most of the variable region of the protein and some portion of the first
constant region domain. Common clinical findings in patients with heavy chain disease are
splenomegaly, hepatomegaly, and lymphadenopathy. Almost all cases of mu chain disease have
been associated with CLL. Gamma chain disease has been found in the presence of a variety
of autoimmune disorders and in lymphoplasmacytic lymphoma. Alpha chain disease is associ-
ated with extranodal marginal zone lymphoma of the MALT type, which usually involves the
gastrointestinal tract.
Diagnosis
The diagnosis of heavy chain disease is made primarily by demonstration of a monoclonal heavy
chain by protein electrophoresis of serum, concentrated urine, or both. The diagnosis of heavy
chain disease should prompt an investigation into the presence of lymphoma if that diagnosis has
not already been made.
Monoclonal Gammopathies of Unknown Significance

Description
Patients with MGUS are asymptomatic but have a monoclonal protein in their serum and/or
urine. There is an increasing incidence of MGUS with aging. Because the incidence of malignant Because the incidence of
monoclonal gammopathies also increases with age, it is essential to differentiate patients who malignant monoclonal
have MGUS from those who have plasma cell myeloma or Waldenström macroglobulinemia. gammopathies also
Most patients with MGUS remain clinically stable without therapy for many years. However, as increases with age, it is
many as 15% to 20% develop myeloma, macroglobulinemia, amyloidosis, or lymphoma. Indo- essential to differentiate
lent myeloma and smoldering myeloma, disorders with many features of multiple myeloma and patients who have
Waldenström macroglobulinemia that do not meet the criteria for diagnosis, can be differentiated MGUS from those
from MGUS because MGUS has a lower amount of immunoglobulin in the serum and a lower who have plasma cell
myeloma or Waldenström
percentage of plasma cells in the bone marrow.
macroglobulinemia.
Diagnosis
MGUS is diagnosed by the presence of a monoclonal serum or urine immunoglobulin at a con-
centration less than that required for a myeloma diagnosis, less than 10% abnormal plasma cells
in the bone marrow, no lytic bone lesions, and no symptoms suggestive of multiple myeloma.
Hodgkin Lymphoma
Hodgkin lymphoma is distinguished from non-Hodgkin lymphoma by the presence of a neoplas- Hodgkin lymphoma
tic giant cell known as a Reed–Sternberg cell in the lymph node. For many years the lineage of is distinguished from
the Reed–Sternberg cell was controversial, but the best information now indicates that the Reed– non-Hodgkin lymphoma
Sternberg cell is an abnormal malignant B cell. Hodgkin disease is a common form of malig- by the presence of a
nancy in young adults with a second peak incidence in older individuals. Unlike the multiple neoplastic giant cell
classification schemes for non-Hodgkin lymphomas, a classification of Hodgkin disease known known as a Reed–
as the Rye classification was accepted for decades. This classification system has now been incor- Sternberg cell in the
porated with minor changes into the WHO classification system for hematologic malignancies. lymph node.
Hodgkin lymphoma is divided into 2 broad categories: classical Hodgkin lymphoma and nodu-
lar lymphocyte-predominant Hodgkin lymphoma. Classical Hodgkin lymphoma is character-
ized by infrequent Reed–Sternberg cells in a background of normal lymphocytes, plasma cells,
eosinophils, and granulocytes. The Reed–Sternberg cells lack expression of the pan-hematopoi-
etic marker CD45; they occasionally express the B-cell marker CD20, and they characteristically
express CD30 and CD15. The different subtypes of classical Hodgkin lymphoma in the WHO
classification are characterized primarily by differences in tissue architecture and the composition
of the cellular background.
Nodular lymphocyte-predominant Hodgkin lymphoma also shows scattered large abnor-
mal cells, but these do not have the appearance of Reed–Sternberg cells. The abnormal cells in
nodular lymphocyte-predominant Hodgkin lymphoma have convoluted nuclei, leading to the

term “popcorn cells.” These abnormal cells express the pan-hematopoietic marker CD45 and the
B-cell marker CD20; they variably express CD30 and do not express CD15. The abnormal cells
are frequently ringed by normal T cells. Nodular lymphocyte-predominant Hodgkin lymphoma
is best thought of as a low-grade B-cell lymphoma.
MYELOID DISORDERS
Acute Myeloid Leukemias
An acute leukemia Acute leukemias are hematologic malignancies that primarily involve the peripheral blood and
is a neoplasm of a bone marrow. An acute leukemia is a neoplasm of a hematopoietic stem cell that has lost its capac-
hematopoietic stem ity to differentiate and regulate its own proliferation. The outcome of the expansion of the leu-
cell that has lost its kemic clone is an accumulation of poorly differentiated WBC precursors known as blasts in the
capacity to differentiate bone marrow. These limit the production of normal blood cells. The blasts commonly appear in
and regulate its own the peripheral blood and permit identification of an acute leukemia from review of the peripheral
proliferation. The blood smear. A diagnosis of acute leukemia requires that 20% or more of the bone marrow cells be
outcome of the expansion blasts. These disorders are usually rapidly progressive, and patients can die within days to weeks
of the leukemic clone
without therapeutic intervention. In the WHO classification, leukemias are not considered an
is an accumulation of
independent group of diseases. Instead, they are classified according to their cell of origin. Acute
poorly differentiated
leukemias of lymphoid cells are included in the B- and T-cell malignancy classification scheme.
WBC precursors known
as blasts in the bone In this section, we will consider the acute myeloid leukemias, which are malignancies of myeloid
marrow. stem and precursor cells.
Leukemias cannot generally be diagnosed by morphology alone. In particular, it is fre-
quently not possible to distinguish leukemias of myeloid lineage from leukemias of B- or T-cell
precursors. Special cytochemical stains can sometimes help identify the lineage of the leukemic
cells, but definitive classification is best done by immunophenotyping using flow cytometry.
It is also important to perform cytogenetic and/or molecular analysis of leukemias, to obtain
prognostic information and, in some cases, information that can be used to design specific
therapy. The description and diagnosis for several acute leukemias are presented in the sections
that follow.
A uniform classification for the acute leukemias was developed in 1976 by the French–
American–British (FAB) cooperative group. The classification from the FAB cooperative group
ultimately divided AML into 7 types, M1 to M7. These types were based primarily on the mor-
phology of the leukemic blasts and in some cases on cytochemical staining. In 1990, the National
Cancer Institute established guidelines for an M0 type of AML that is not within the M1 to M7
classification. The distinction between AML and the myelodysplastic syndromes was clarified at
that time (see the section “Myelodysplastic Syndromes”). As more has been learned about the
molecular pathophysiology of AML, it has become clear that the FAB categories do not corre-
spond to distinct biological entities. For example, the translocation t(8;21) can be found in several
different FAB types. The one exception is FAB type M3, acute promyelocytic leukemia. This leu-
kemia reproducibly has a translocation t(15;17), and is unique among the myeloid leukemias in
its response to treatment by all-trans retinoic acid or arsenic trioxide.
The 2008 WHO classification system recognizes some of the drawbacks of the FAB sys-
tem. New features of the WHO system include defining specific leukemias by their molecular
pathology for those with recurrent chromosomal abnormalities, and creating categories for
leukemias evolving from previous myelodysplastic syndromes and from patients treated with
leukemogenic chemotherapy for prior malignancies that do not fit neatly into the FAB categories
(see Table 13–3). For the remaining myeloid leukemias (“not otherwise specified”), classification
is similar to the FAB system (see Table 13–4).
Leukemias With Recurrent Genetic Abnormalities

Certain common chromosomal translocations are found in acute myeloid leukemia. These
translocations usually result in the generation of an abnormal transcription factor that alters
gene expression, leading to leukemia. The most common recurrent translocation is the t(8;21)
(q22;q22) involving the genes RUNX1–RUNX1T1. It is seen in several FAB types of AML and is
TABLE 13–3 2008 World Health Organization Classification of Myeloid Neoplasms

Acute myeloid leukemias
Acute myeloid leukemias with recurrent cytogenetic abnormalities

AML with t(8;21)(q22;q22), RUNX1/RUNX1T1
AML with inv(16) or t(16;16)(p13;q22), CBFB/MYH11
Acute promyelocytic leukemia (AML) with t(15;17)(q22;q12), PML/RARA, and variants
AML with t(9;11)(p22;q23), MLLT3/MLL
AML with t(6;9)(p32;q34), DEK/NUP214
AML with inv(3)(q21;q26.2) or t(3;3)(q21;q26.2), RPN1/EVI1
AML (megakaryoblastic) with t(1;22)(p13;q13), RBM15/MKL1
Acute myeloid leukemia with myelodysplasia-related changes

Acute myeloid leukemia, therapy-related
Acute myeloid leukemia, not otherwise characterized
AML minimally differentiated
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic and monocytic leukemia
Acute erythroid leukemia
Acute megakaryoblastic leukemia
Acute leukemias of ambiguous lineage
Myeloproliferative neoplasms
Chronic myeloid leukemia

Chronic neutrophilic leukemia
Polycythemia vera
Primary myelofibrosis
Essential thrombocythemia
Chronic eosinophilic leukemia
Mastocytosis
Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1
Myelodysplastic/myeloproliferative neoplasms
Chronic myelomonocytic leukemia
Atypical chronic myeloid leukemia
Juvenile myelomonocytic leukemia
Myelodysplastic syndromes
Refractory cytopenia with unilineage dysplasia (anemia, neutropenia, or thrombocytopenia)
Refractory anemia with ringed sideroblasts
Refractory cytopenia with multilineage dysplasia
Refractory anemia with excess blasts
MDS with isolated del(5q) chromosome abnormality
Data from Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.
Lyon, France: IARC Press; 2008.
associated with a relatively good prognosis. The inv(16) translocation involves the genes CBFB–
MYH11 and is associated with a type of myelomonocytic leukemia that has increased eosinophils
in the bone marrow (FAB type AML M4-Eo). It is also associated with a relatively good progno-
sis. The t(15;17) translocation involves the genes PML–RARA and is uniquely associated with
acute promyelocytic leukemia (FAB type M3). This translocation results in an abnormal form of
the retinoic acid receptor α (RARA). Interestingly, acute promyelocytic leukemia can be treated Acute promyelocytic
with all-trans retinoic acid in addition to standard leukemia chemotherapy, and many patients leukemia can be treated
have a good outcome. The all-trans retinoic acid presumably interacts with the abnormal prod- with all-trans retinoic acid
uct of the t(15;17) fusion gene and interferes with its leukemogenic function. This was the first in addition to standard
acute leukemia therapy described that was directed at the molecular pathology of the leukemia. leukemia chemotherapy,
Another recurrent chromosomal abnormality associated with AML is the t(9;11) involving the and many patients have a
genes MLLT3–MLL, the latter of which is at chromosome 11q23. In addition to MLLT3, MLL good outcome.
forms fusion genes with many different partner genes. It is found more commonly in pediatric
AML and is associated with a somewhat worse clinical outcome. Rarer translocations that are
defined entities in the WHO classification include t(6;9)/DEK–NUP214, inv(3)/RPN1–EVI1, and
t(1;22)/RBM15–MKL1.
TABLE 13–4 Classification of Acute Myeloid Leukemias, Not Otherwise Specified

Blast Corresponds
Percentage Cytochemistry Immunophenotype Genetics Other to FAB Type
AML minimally ≥20 MPO and NSE CD13, CD33, CD117, 27% with M0
differentiated negative usually CD34, CD38, RUNX1T1
HLA-DR; may have mutations;
low expression of 16%-22%
lymphoid markers with FLT3
TdT, CD7, CD2, mutations
CD19, no monocytic
markers
AML without ≥90 MPO positive CD13, CD33, CD117, No specific M1
maturation >3%, NSE MPO, frequently abnormalities
negative CD34 and HLA-DR,
generally negative
for monocytic and
lymphoid markers
AML with 20-89 MPO positive, CD13, CD33, CD15, No specific Less than 20% M2
maturation NSE negative may express CD117, abnormalities monocytes in
CD34, HLA-DR, marrow
20%-30% with CD7
Acute 20-89 MPO and NSE CD13, CD15, CD33, No specific Greater than or M4
myelomonocytic positive HLA-DR, positive abnormalities equal to 20%
leukemia for some monocytic monocytes in
antigens (including marrow, may
CD14, CD4, CD11b, have peripheral
CD11c, CD64, CD36, monocytosis
lysozyme), 30% have
CD7, subset may
have CD34, CD117
Acute 20-89 MPO negative, CD13, CD33 (bright), No specific Greater than M5a
monoblastic/ NSE positive HLA-DR, monocyte abnormalities; 80% monoblasts (monoblastic),
monocytic markers (including occasional or monocytes M5b
leukemia CD14, CD4, CD11b, t(8;16) in marrow: if (monocytic)
CD11c, CD64, CD36, monoblasts,
lysozyme), frequently then acute
have CD7, CD56 monoblastic
leukemia; if
promonocytes,
then acute
monocytic
leukemia
Acute erythroid Erythroleukemia MPO, NSE CD13, CD33, CD117, Complex M6

leukemia has ≥50% negative; PAS may have MPO, cytogenetics,
(erythroleukemia total marrow positive CD34, HLA-DR, no specific
and pure erythroid erythroid cells abnormalities
erythroid cells, ≥20% of express glycophorin
leukemia) nonerythroid A, hemoglobin
cells are blasts;
pure erythroid
leukemia has
≥80% immature
erythroid cells
Acute 20-89 MPO, NSE Platelet markers: Complex Abnormal M7

megakaryoblastic negative CD41, CD61, less cytogenetics, megakaryocytes
leukemia CD42, some with no specific in marrow,
CD13, CD33, CD36, abnormalities frequent
often negative for marrow fibrosis
CD34, CD45, HLA-DR
Data from Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008.
Secondary Acute Myeloid Leukemia

Unlike the leukemias with recurrent chromosomal abnormalities, which are thought to represent
de novo events, there is a group of leukemias that evolve out of previously existing conditions.
One set consists of the leukemias that develop from patients with stem cell disorders such as
the myelodysplastic syndromes (see below). These leukemias are associated with morphological
abnormalities in all hematopoietic lineages. The other secondary leukemias develop in patients
who have had previous chemotherapy for other malignancies. These leukemias occur in patients
who were treated with alkylating agents such as cyclophosphamide or nitrogen mustard or with
topoisomerase inhibitors such as the epipodophyllotoxins or anthracyclines. Both sets of leuke-
mias are associated with complex cytogenetic abnormalities and have poor prognoses.
Other Acute Myeloid Leukemias

Leukemias that do not have characteristic cytogenetic abnormalities or documented previous
stem cell disorders or therapy are characterized by their putative lineage as determined by immu-
nophenotyping, morphology, and cytochemical staining. Their classification is most similar to the
FAB system used prior to the WHO classification. The diagnostic criteria for these leukemias are
shown in Table 13–4.
Biphenotypic and Mixed Lineage Leukemias

There is a subset of acute leukemias that express both myeloid and lymphoid markers at the same
time on the same blasts. These are called mixed lineage leukemias. These leukemias may reflect
a lack of marker specificity or aberrant gene expression by a malignant hematopoietic stem cell.
Biphenotypic leukemias have separate subpopulations of leukemic blasts with different immuno-
phenotypes (eg, myeloid on 1 set and lymphoid on another). Both types of leukemias generally
have a relatively poor prognosis.
Diagnostic Techniques for AML

• Cytochemistry—Two cytochemical stains are widely used in the diagnostic evaluation of MPO identifies cells
an acute leukemia. These are myeloperoxidase (MPO) and nonspecific esterase (NSE). of myeloid lineage,
MPO identifies cells of myeloid lineage, which usually stain intensely positive for MPO. which usually stain
Monoblasts and promonocytes, which appear in acute myelomonocytic leukemia, can also intensely positive
react with MPO. NSE is confined mostly to cells of monocytic lineage, which predominate for MPO. Monoblasts
in acute monoblastic/monocytic leukemia. and promonocytes,
• Immunophenotyping—With the development of monoclonal antibodies that can be which appear in acute
fluorochrome-conjugated to bind to and identify cell antigens, flow cytometry has myelomonocytic
become a useful tool in distinguishing AML from ALL, and identifying the individual leukemia, can also
react with MPO. NSE is
subtypes of AML. Immunophenotyping is particularly important in the identification
confined mostly to cells of
of blasts that show no morphological features to indicate their lineage, as found in the
monocytic lineage, which
minimally differentiated subtype of AML. Markers such as CD14 and CD64 can be useful predominate in acute
in identification of monocytic cells in AML. The detection of hemoglobin or glycophorin monoblastic/monocytic
A aids in the diagnosis of erythroleukemia. Identification of platelet glycoprotein antigens leukemia.
supports a diagnosis of acute megakaryoblastic leukemia.
• Cytogenetics and FISH—As described above, certain types of AML are defined by specific
chromosomal rearrangements. Traditionally, chromosomal rearrangements are detected by
cytogenetic studies, in which the chromosomes of dividing leukemic blasts are stained and
examined under the microscope, which allows the different chromosomes to be identified,
and where abnormal chromosome rearrangements can be detected. To look for specific
gene rearrangements, FISH is used, which is the fastest and most sensitive method for
detecting the specific rearrangements in leukemias with recurrent genetic abnormalities.
• Molecular genetics—Molecular genetic tests are also used to provide diagnostic and/or
prognostic information not available from morphological analysis. For example, PCR
gene amplification (in addition to FISH) can be used to detect the recurrent cytogenetic
translocations of AML. Molecular analysis can also be used to detect mutations in
specific genes such as FLT3, NPM1, and CEBPA, which frequently occur in AML with
otherwise normal cytogenetics. Finally, PCR to detect translocation-specific fusion RNAs

can sensitively detect residual leukemic cells after chemotherapy or transplantation and
thereby permit earlier intervention.
Laboratory Approaches for Determination of AML Prognosis

Acute myeloid leukemia is currently treated initially with one of a small number of chemotherapy
regimens, all of which are quite toxic to the bone marrow and other organs. By studying large num-
bers of patients with AML, it has become clear that certain types of AML tend to respond well to
standard chemotherapy, whereas others do poorly and require more intensive chemotherapy regi-
mens and/or stem cell transplantation if a patient has a chance to be cured. This research has shown
that cytogenetic and molecular abnormalities correlate well with prognosis, and thus the clinical
laboratory has a critical role to play in determining the therapy for patients with AML: the goal is
to identify those patients who need more aggressive treatment at the time of their diagnosis while
sparing those with a better prognosis exposure to unnecessary and possibly life-threatening therapy.
The traditional way The traditional way of determining AML prognosis has been to use cytogenetic studies to
of determining AML place patients into favorable, unfavorable, or intermediate risk groups (see Table 13–5). Favorable
prognosis has been to risk cytogenetics include AML with t(8;21), inv(16), or t(15;17). Unfavorable risk cytogenetics
use cytogenetic studies include AML lacking more than 1 chromosome, inv(3), MLL rearrangements, and AML with
to place patients into multiple cytogenetic abnormalities. Those meeting neither favorable risk nor unfavorable risk
favorable, unfavorable, or criteria are considered intermediate risk, including AML with no cytogenetic abnormalities.
intermediate risk groups. Further information about AML prognosis has come from studying individual genes found
Further information about
to have an impact on a patient’s likelihood of cure with standard chemotherapy. One of the most
AML prognosis has come
important genes for prognosis is the FLT3 gene. When this gene is mutated in AML cells, it puts
from studying individual
a patient into an unfavorable risk category, even if the AML has other more favorable risk charac-
genes found to have an
impact on a patient’s teristics. In contrast, mutation of the NPM1 gene confers more favorable risk if there is no FLT3
likelihood of cure with mutation. Similarly, if both copies of the CEBPA gene are mutated, the patient is considered favor-
standard chemotherapy. able risk, again in the absence of a FLT3 mutation. Mutation of the KIT gene confers a poor prog-
nosis on the t(8;12) and inv(16) AMLs, which ordinarily have a good prognosis. Currently many
more genes are being recognized as having an impact on AML prognosis, and genetic analysis
is likely to replace cytogenetics for determining AML prognosis. Next-generation sequencing
technologies are rapidly evolving and will allow numerous genes, or even the entire genome, of
AML cells to be determined, which will improve the ability to determine disease prognosis and
hopefully lead to therapies that can target each individual’s unique cancer cells.
TABLE 13–5 Prognostic Markers in Acute Myeloid Leukemia

Good prognosis
AML with t(8;21)(q22;q22), RUNX1/RUNX1T1

AML with inv(16) or t(16;16)(p13;q22), CBFB/MYH11
Acute promyelocytic leukemia (AML) with t(15;17)(q22;q12), PML/RARA, and variants
NPM1 mutation without FLT3 mutation
Biallelic CEBPA mutation without FLT3 mutation
Intermediate prognosis
Normal karyotype
Neither good nor poor prognosis
FLT3 mutation with NPM1 mutation
FLT3 mutation with CEBPA mutation
Poor prognosis
AML with t(9;11)(p22;q23), MLLT3/MLL, and other MLL translocations

AML with t(6;9)(p32;q34), DEK/NUP214
AML with inv(3)(q21;q26.2) or t(3;3)(q21;q26.2), RPN1/EVI1
AML with t(9;22)(q34;q11), BCR/ABL1
Monosomal karyotype (multiple chromosome losses)
Complex karyotype (more than 4 abnormalities)
FLT3 mutation without other modifying mutations
TP53 mutations
MYELOPROLIFERATIVE NEOPLASMS
Disorders in which there is a clonal neoplastic proliferation of a multipotent myeloid stem cell are Disorders in which there
grouped together in the myeloproliferative disorders. The major disorders in this grouping include: is a clonal neoplastic
proliferation of a
• Chronic myeloid leukemia, with cell proliferation in the granulocytic series multipotent myeloid stem
• Polycythemia vera, in which erythrocytic precursors dominate the picture (see the section cell are grouped together
“Erythrocytosis” in Chapter 10) in the myeloproliferative
• Essential thrombocythemia in which megakaryocytes are the primary cytological feature disorders.
(see the section “Bleeding Disorders” in Chapter 11)
• Primary myelofibrosis (see the following), a disorder in which the bone marrow is initially
hypercellular in multiple cell lineages and then gradually becomes markedly hypocellular
with the development of marrow fibrosis
The myeloproliferative neoplasms have a fair amount of overlap in their clinical and hema-
tologic findings, including increased numbers of red cells, platelets, and/or white cells, the pres-
ence of circulating immature cells, and the presence of marrow fibrosis. The fibrosis is a reactive
response to the neoplastic elements of the bone marrow. Myeloproliferative neoplasms are differ-
entiated from the myelodysplastic disorders because in the myeloproliferative neoplasms, there
are few, if any, dysplastic changes in the blood cell precursors in the marrow. The prognosis of the
myeloproliferative neoplasms varies depending on the diagnosis. Polycythemia vera and essential
thrombocythemia tend to have very long survival and a low incidence of transformation to acute
leukemia. Chronic myeloid leukemia and primary myelofibrosis have worse prognoses.
Chronic myeloid leukemia is distinct from the other myeloproliferative disorders in that it
contains a specific chromosomal translocation, t(9;22)(q34;q11), also known as the Philadelphia
chromosome. This will be discussed in detail below.
The other myeloproliferative disorders commonly contain a mutation in the JAK2 tyrosine
kinase gene. JAK2 is a tyrosine kinase involved in transmitting growth signals for several different
hematopoietic growth factors. In approximately 80% to 90% of polycythemia and in approxi-
mately 40% to 50% of essential thrombocythemia and primary myelofibrosis, the valine at amino
acid 617 is mutated to a phenylalanine (designated V617F). This mutation inactivates a domain
of the JAK2 protein that normally inhibits its tyrosine kinase activity. The result is that the kinase
becomes activated without growth factor stimulation, and this leads to uncontrolled proliferation
of the cells. Molecular testing for the V617F mutation has become standard practice for patients
suspected of having a myeloproliferative neoplasm.
Chronic Myeloid Leukemia

Description
CML represents approximately 15% of all leukemias in the United States. The median age at diag-
nosis is 65 years, and there is a slight male predominance, with a male to female ratio of 1.7:1. In
CML, the disease begins with a chronic phase that usually lasts for 3 to 4 years after diagnosis. The
chronic phase evolves into a more aggressive accelerated phase of the disease. This phase persists
for 1 to 2 years in most cases. At least 25% of patients with CML die in this phase of the disease.
The remainder progress to an acute leukemia, which is known as blast crisis. The blast crisis typi-
cally leads to death within 6 months because it is highly resistant to chemotherapy. Approximately
25% of the patients with CML advance rapidly from chronic phase to blast crisis, without a sig-
nificant intervening period of acceleration. Hematopoietic stem cell transplantation in chronic
phase for patients who can tolerate the procedure is highly effective at curing CML.
CML is characterized by a characteristic chromosomal translocation, t(9;22), also known CML is characterized
as the Philadelphia chromosome. Discovered in 1960, this was the first genetic lesion associ- by a characteristic
ated with a human cancer. When molecular cloning techniques became available, the t(9;22) was chromosomal
found to produce an abnormal RNA and protein product, BCR–ABL1. BCR–ABL1 is a tyrosine translocation, t(9;22), also
kinase that is constitutively active and leads to uncontrolled proliferation of myeloid cells. In 1996, known as the Philadelphia
a drug, imatinib mesylate, was discovered that inhibits the tyrosine kinase activity of BCR–ABL1, chromosome.
and this has led to a revolution in the treatment of CML, which previously relied primarily on
interferon-alpha and bone marrow transplantation. Long-term treatment with imatinib can lead
to drug resistance however, and new BCR–ABL1 inhibitors are in development to overcome this
problem. The only curative treatment for CML is still bone marrow transplantation.
Diagnosis
The diagnosis of CML is based on the morphological appearance of the bone marrow, periph-
eral blood cell morphology, and cytogenetic and molecular genetic studies. Cytochemistry and
immunophenotyping are not particularly valuable in the diagnosis of CML in its chronic phase.
This is the stage of the disease in which most CML patients first present.
• Chronic-phase CML—A significant hematologic finding in this phase of CML is a
moderate to significant elevation of the neutrophil count, often with all stages of neutrophil
maturation detectable in the peripheral blood smear. An increase in basophils is important
to recognize, as modest basophilia is an early indication of CML. Approximately 50% of
CML patients also have an elevation in their platelet count. The appearance of the bone
marrow is hypercellular as the disease progresses in the chronic phase of CML, with
an increase in the myeloid/erythroid ratio from 2:1-4:1 to 10:1-30:1. There is complete
maturation of the granulocytes in CML.
• Accelerated-phase CML—There is no widely accepted definition for the accelerated phase
of CML. The characteristic features of this phase of the disease include splenomegaly,
an increase in the proportion of myeloblasts (10%-19%) and promyelocytes in the bone
marrow over that found in the chronic phase, basophilia to >20% of the total WBC count,
and anemia or thrombocytopenia.
• CML in blast crisis—By definition, when the percentage of blasts is 20% or more in the
blood or bone marrow, blast transformation of CML has occurred. The blasts can be
of either myeloid or lymphoid lineage; this determination is made by flow cytometry
immunophenotyping. Approximately 70% of blast crises are myeloid. CML transforms
into ALL in approximately 30% of cases of blast crisis. The immunophenotype is most
commonly precursor B-cell ALL.
• Cytogenetics—The Philadelphia chromosome, t(9;22), is present in essentially 100% of
CML cases; if the Philadelphia chromosome cannot be demonstrated by cytogenetics,
FISH, or molecular studies, another diagnosis should be considered. Blast crisis is usually
accompanied by additional cytogenetic abnormalities that appear with clonal evolution.
• Molecular genetics—The diagnosis of CML is still possible in cases that are Philadelphia
chromosome negative by using FISH or reverse transcriptase PCR to detect the BCR–ABL1
fusion RNA. PCR can also be used to detect minimal residual disease in patients being
treated for CML. Newer techniques are now available to quantify BCR–ABL1 RNA in
the peripheral blood or bone marrow. These are being used to assess clinical responses to
imatinib and to detect early evidence of imatinib resistance.
Polycythemia Vera
Description
Polycythemia vera is Polycythemia vera is diagnosed by an increase in red cell mass with no apparent cause such as
diagnosed by an increase chronic oxygen deprivation (living at high altitude or heavy smoker). Transformation to acute
in red cell mass with myeloid leukemia is rare, but patients with polycythemia vera are at increased risk for the devel-
no apparent cause opment of leukemia (see Chapter 10 for additional information on polycythemia vera).
such as chronic oxygen
deprivation (living at high
Diagnosis
altitude or heavy smoker).
• Cell counts and the peripheral blood smear—By definition, the hemoglobin, hematocrit,
and red cell count are all elevated. Patients often present with microcytosis and a normal
hematocrit due to the iron deficiency that develops due to excessive red cell production.
Because of this, these patients are sometimes initially thought to have thalassemia trait. The
WBC count and platelet count are also often moderately elevated.
• Bone marrow morphology—The bone marrow can appear normal, but is often hypercellular
with an increase in red cell precursors. With progressive disease, the bone marrow can
become fibrotic.
• Cytogenetics and molecular pathology—Cytogenetic findings are usually normal. Definitive

diagnosis is made by demonstrating a point mutation in the JAK2 gene.
Essential Thrombocythemia
Description
Essential thrombocythemia is diagnosed by an increase in platelet count with no other explana-
tion. Platelet counts can frequently exceed 1 million/μL. Patients with essential thrombocythemia
can manifest abnormal bleeding or blood clotting, although these complications are not very
common. Transformation to acute myeloid leukemia is rare (see Chapter 11 for additional infor-
mation on essential thrombocythemia).
Diagnosis
• Cell counts and the peripheral blood smear—Diagnosis is made by demonstrating a chronically
elevated platelet count. The WBC count and hematocrit may also be moderately elevated.
• Bone marrow morphology—The bone marrow demonstrates an increase in megakaryocytes
and an overall increase in cellularity. With progressive disease, the bone marrow can
become fibrotic.
• Cytogenetics and molecular pathology—Cytogenetic findings are usually normal. Definitive
diagnosis is made by demonstrating a point mutation in the JAK2 gene, which occurs in
about 50% of cases. Activating mutations in the thrombopoietin receptor gene, MPL, have
also been described in 5% to 10% of cases.
Primary Myelofibrosis
Description
Patients with primary myelofibrosis typically present with marked splenomegaly and some degree
of hepatomegaly. The disease affects primarily older individuals. As the marrow becomes fibrotic
and cytopenias in the peripheral blood develop, the complications associated with the cytopenias
appear. Bleeding from low platelet counts and infections from low WBC counts may be lethal. A
minority of patients with primary myelofibrosis (less than 10%) progress to acute leukemia, with
a higher incidence in those who are treated with radioactive phosphorus or alkylating agents in
the highly proliferative phase of their disease.
Diagnosis
• Cell counts and the peripheral blood smear—The peripheral blood frequently demonstrates
a “leukoerythroblastic” picture, with leukocytosis, immature granulocytes including blasts,
thrombocytosis, and the presence of immature erythroid cells including reticulocytes and
nucleated red cells. The cell counts decline with disease progression.
• Bone marrow morphology—Initially, the bone marrow shows trilineage hypercellularity
with megakaryocyte hyperplasia and reticulin fibrosis. With disease progression, there Myelodysplastic
is replacement of the bone marrow by extensive fibrosis allowing little space for hematopoiesis. syndromes include a
• Cytogenetics and molecular pathology—Cytogenetic abnormalities are present in 30% to group of bone marrow
60% of patients. Approximately 40% to 50% of patients with idiopathic myelofibrosis have disorders with dysplastic
the JAK2 V617F mutation. (not normal, but not
neoplastic) changes of the
cells of the myeloid series.
MYELODYSPLASTIC SYNDROMES In myelodysplasia, the
myeloblasts in the bone
Description
marrow must represent
Myelodysplastic syndromes include a group of bone marrow disorders with dysplastic (not normal, less than 20% of all
but not neoplastic) changes of the cells of the myeloid series. In myelodysplasia, the myeloblasts in nucleated marrow cells,
the bone marrow must represent less than 20% of all nucleated marrow cells, because if there are because if there are 20%
20% or more blasts, a diagnosis of acute myeloid leukemia is made. Because the myelodysplastic or more blasts, a diagnosis
cells originate from an abnormal stem cell clone that is genetically unstable, there is a tendency for of acute myeloid leukemia
myelodysplasia to evolve into acute leukemia. is made.
Many names have been Myelodysplastic syndrome can occur as a primary disease or as a secondary disorder follow-
applied to what is now ing exposure to chemotherapeutic agents or radiotherapy. Most cases of primary myelodysplastic
called myelodysplastic syndrome are found in individuals over the age of 50 years. Many names have been applied to
syndrome, including what is now called myelodysplastic syndrome, including preleukemia, refractory anemia (RA),
preleukemia, refractory and smoldering leukemia.
anemia (RA), and
smoldering leukemia. Diagnosis
Peripheral blood cytopenias are a hallmark of the myelodysplastic syndrome. As a result of inef-
fective hematopoiesis, myelodysplasia patients present with the complications of reduced blood
cell counts in 1 or more cell lines. The complications include infections from low WBC counts,
hemorrhage from low platelet counts, and weakness from anemia. In all forms of myelodysplastic
syndrome, the bone marrow biopsy reveals hypercellularity.
A cytogenetic abnormality is found in 40% to 80% of the cases of primary myelodysplasia
and 90% to 97% of patients with secondary myelodysplasia. These abnormalities may be useful as
prognostic indicators. The most common changes are an interstitial deletion of the long arm of
chromosome 5 (5q–) and deletions of chromosome 7 (–7, 7p–, or 7q–).
WHO Classification of the Myelodysplastic Syndrome

The myelodysplastic syndromes include a heterogeneous, but definable group of disorders. A brief
description of each the disorders in the myelodysplastic syndrome is provided as follows:
• RA—This is defined as an anemia refractory to therapy. Dysplastic changes are only seen in
the erythroid lineage. There are less than 5% bone marrow blasts, and ringed sideroblasts
are less than 15% of the erythroid precursors.
• Refractory anemia with ringed sideroblasts (RARS)—This disorder is like RA except that
15% or more of the nucleated RBCs in the marrow are ringed sideroblasts. A ringed
sideroblast, as noted in the section “Sideroblastic Anemia” (Chapter 10), is a cell in which
at least 30% of the circumference of the nuclear membrane is covered by mitochondria
containing iron granules.
• Refractory cytopenias with multilineage dysplasia (RCMD)—This disorder is like RA except
that dysplasia is present in 2 or more lineages (lineages being myeloid, erythroid, and
megakaryocytic). Auer rods (abnormal inclusions in myeloid blasts) are absent.
• Refractory cytopenias with multilineage dysplasia and ringed sideroblasts (RCMD-RS)—This
disorder is like RCMD except that 15% or more of the nucleated RBCs in the marrow are
ringed sideroblasts.
• Refractory anemia with excess blasts-1 (RAEB-1)—The major criterion for RAEB-1 is
the presence of 5% to 9% of total nucleated cells in the bone marrow as blasts. There can
be unilineage or multilineage dysplasia. In addition, the percentage of WBC blasts in the
peripheral blood must be less than 5% of nucleated cells. Auer rods are absent.
• Refractory anemia with excess blasts-2 (RAEB-2)—This disease is present if there are 10% to
19% blasts in the bone marrow, 5% to 19% blasts in the blood, or Auer rods in myeloblasts
or other neutrophilic precursors. There can be unilineage or multilineage dysplasia.
• Myelodysplastic syndrome, unclassified (MDS-U)—This disease is similar to RA except that
dysplasia is present in 1 lineage other than the erythroid lineage. There are less than 5%
blasts, and there are no Auer rods.
• MDS associated with del(5q)—Also known as “5q- syndrome,” there are normal to increased
megakaryocytes with hypolobated nuclei, less than 5% blasts, no Auer rods, and the sole
cytogenetic abnormality of del(5q).
MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASMS
The myelodysplastic/myeloproliferative neoplasm category was created for the 2008 WHO
classification. This category is for clonal stem cell disorders that do not fit well into either the
myelodysplastic or myeloproliferative disorders. The most common of these disorders is chronic
myelomonocytic leukemia (CMML). Other rare disorders in this category include atypical
chronic myeloid leukemia, juvenile myelomonocytic leukemia, and an unclassifiable category.
• CMML—This disorder is a chronic leukemia with dysplastic changes in the bone marrow that
are indicative of an increased risk for transformation into acute myeloblastic leukemia. Patients
with CMML have an absolute peripheral monocytosis greater than 1000 monocytes/μL, less
than 20% blasts in the bone marrow, and dysplasia of 1 or more myeloid lineages.
DISORDERS ASSOCIATED WITH IMPAIRED WBC FUNCTION

WBCs must be present in appropriate numbers and also function normally. The disorders previ- Many WBC qualitative
ously discussed in this chapter are associated with alterations in WBC number, and in some cases, disorders result in
impaired function as well. The 3 disorders presented in the following sections represent examples functional impairment
of WBC functional disorders with no alteration in WBC number. It is for this reason that they are and no increased risk
also known as qualitative (as opposed to quantitative) WBC disorders. Many WBC qualitative dis- for infections. However,
orders result in functional impairment and no increased risk for infections. However, other WBC other WBC functional
functional abnormalities may be clinically significant and predispose to life-threatening infections. abnormalities may be
clinically significant
and predispose to life-
Chediak–Higashi Syndrome threatening infections.
Description
This disorder is due to a mutation in a lysosomal trafficking regulator. The disorder is charac-
terized by functional defects associated with azurophilic granules in the cells that have these
granules. They are particularly prominent in neutrophils and melanocytes. Most patients with
Chediak–Higashi syndrome are subject to recurrent infections. Chediak–Higashi patients also
have partial albinism because they have defective melanosomes (which provide skin coloration).
The platelets from these patients have a defect in storage granules. This platelet granule deficiency
may produce a bleeding tendency because release of the granule contents is necessary for the
platelets to aggregate and form platelet plugs.
Diagnosis
A personal and family history consistent with Chediak–Higashi syndrome, along with abnormal
granules in all granulated hematopoietic cells and lymphocytes, strongly suggests the diagnosis.
Chronic Granulomatous Disease

Description
Chronic granulomatous disease (CGD) comprises a heterogeneous group of disorders in which
recurrent bacterial infections can lead to an early death. The WBCs in CGD do not exhibit obvi-
ous morphological differences from normal WBCs. However, there are multiple biochemical
defects in neutrophil function in CGD that limit their ability to produce peroxide and superox-
ides that destroy bacteria.
Diagnosis
Patients with CGD have a negative nitroblue tetrazolium (NBT) dye test. In this assay, a yel-
low dye is oxidized by the oxidative enzymes in the normal granules of neutrophils to form an
insoluble blue-black compound detectable by light microscopy.
Myeloperoxidase Deficiency
Description
This disorder results from a defect in the pathway required for generation of free radicals, which
are important in the destruction of invading microorganisms, similar to CGD. Although indi-
viduals with MPO deficiency may experience recurrent infections, the disorder is benign in most
cases. The absence of MPO may be congenital or acquired.
Diagnosis
In patients with MPO deficiency, MPO staining of freshly prepared blood smears will produce
only faint staining of the granules in neutrophils.
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C H A P T E R
The Respiratory System

Alison Woodworth 14
LEARNING OBJECTIVES
1. Learn the role of blood gases in the evaluation of the patient with pulmonary
disease.
2. Understand how pleural fluid analysis is used in the diagnosis of pulmonary
disorders.
3. Understand how bronchoalveolar lavage can aid in the diagnosis of
respiratory disease.
4. Recognize the different laboratory tests used for assessing fetal lung maturity.
CHAPTER OUTLINE
Approach to the Patient With Pulmonary Asthma 346
Disease 339 Chronic Obstructive Pulmonary Disease 346
Blood Gases and Blood pH 342 Respiratory Distress Syndrome 347
Electrolytes and Anion Gap 342 Selected Laboratory Tests for Assessment
Pleural Fluid Analysis 343 of Fetal Lung Maturity 347
Bronchoalveolar Lavage Fluid Analysis 345 Lung Cancer 348
Laboratory Tests Useful in the Diagnosis or
Management of Pulmonary Diseases 345
Common Lung Disorders 346
APPROACH TO THE PATIENT WITH PULMONARY DISEASE

Impaired exchange of gases is the unifying theme of respiratory disorders (Figure 14–1).
Although they do not play a significant role in the diagnosis of specific pulmonary diseases, blood
gas and electrolyte measurements (see Chapter 2 for blood gas determinations) are commonly
used to assess the severity of different pulmonary abnormalities. The analysis of fluid located in
the pleural space, which collects with certain abnormalities, constitutes another battery of tests
often useful in the evaluation of pulmonary diseases. Careful analysis of respiratory secretions
along with accompanying white blood cells, immune mediators, and foreign pathogens through
bronchoalveolar lavage (BAL) aids in the diagnosis and management of lung disease. Infections in
the respiratory tract are a major cause of pulmonary disorders. These are nearly always diagnosed
using clinical laboratory tests (respiratory infections are discussed in Chapter 5). This chapter
begins with a discussion of laboratory tests utilized in the diagnosis and monitoring of respiratory
diseases and is followed by a section describing the most common lung diseases—asthma, respi-
ratory distress syndrome (RDS) in adults and neonates, chronic obstructive pulmonary disease
(COPD), and lung cancer.
Pulmonary disorders other than infections and tumors can be classified into 3 major
groups. One group of disorders includes emphysema, bronchitis, asthma, and RDS. These are
airway-based (bronchial) diseases. Inflammation or damage to the bronchi results in impaired
339
340 CHAPTER 14 The Respiratory System
Major causes of respiratory disease
Pulmonary
Interstitial
vasculature
lung disease
Airway disease disease
(ie, sarcoidosis,
Tumors Infections (ie, asthma and (ie, pulmonary
pneumoconiosis,
COPD) embolism and
and many
pulmonary
others)
hypertension)
FIGURE 14–1 An overview of the major causes of respiratory disease.
ventilation of alveoli (Figure 14–2). Another group of disorders affects the blood vessels, and,
thereby, blood flow in the lung. Pulmonary vascular diseases are associated with reduced blood
flow, resulting in nonuniform perfusion of the lungs (Figure 14–3). The third major group of
pulmonary diseases is the interstitial lung disorders, which affect the tissue and space around the
alveoli. These are a heterogeneous group of disorders associated with lung tissue damage due to
scarring or inflammation. Damaged lung tissue is unable to fully expand, leading to thickening
of the interface between alveoli and adjacent capillaries resulting in impaired diffusion of gases
(Figure 14–4).
Clinical progression of pulmonary disease can lead to life-threatening respiratory failure.
Acute RDS is a rapid-onset disease associated with severe breathing problems resulting from mul-
tiple insults to the lung (see more information below). It is distinct from “end-stage lung,” which
is the result of a chronic pulmonary disorder.
Normal airway
with no Obstructed
obstruction airway
Alveoli
Blood
vessels
FIGURE 14–2 Diagram of diseases of the airway resulting in impaired ventilation.

CHAPTER 14 The Respiratory System 341
Alveoli
Blood vessel Blood Normal

with pulmonary vessels blood vessel
embolus with no
obstruction
FIGURE 14–3 Diagram of diseases with reduced blood flow, resulting in impaired
perfusion of the lung.
Normal non-thickened Thickened

alveolar-capillary alveolar-capillary
barrier barrier with impaired
gas transfer
Alveoli
Blood
vessels
FIGURE 14–4 Diagram of diseases with a thickened interface between alveoli and adjacent
capillaries, resulting in impaired diffusion of gases.
BLOOD GASES AND BLOOD pH

Many disorders are associated with abnormalities in arterial blood po2, pco2, and pH. While these
tests are not themselves diagnostic, they are valuable in assessment of the severity of respiratory
diseases. The blood gas test panel includes:
• po2 or partial pressure of oxygen
• pco2 or partial pressure of carbon dioxide
• pH
The functional abnormalities most commonly detected with this battery of tests are:
• Low po2 (hypoxemia or low O2 in blood, as opposed to hypoxia, which is reduced O2
in tissues)
• High pco2 (hypercapnia)
• Low arterial blood pH with a primary respiratory cause (respiratory acidosis from an
The lungs respond increased arterial pco2)
within minutes to • Low arterial blood pH with a primary metabolic cause (metabolic acidosis, usually from
acid–base disturbances increased acid production and/or impaired renal H+ elimination, resulting in a decreased
by 1) eliminating CO2 arterial HCO3−)
(hyperventilation) • High arterial blood pH with a primary respiratory cause (respiratory alkalosis from
to increase blood pH a decreased arterial pco2)
or 2) retaining CO2 • High arterial blood pH with a primary metabolic cause (metabolic alkalosis from an
(hypoventilation) to increased arterial HCO3−)
decrease the pH.
The lungs respond within minutes to acid–base disturbances by 1) eliminating CO2 (hyper-
ventilation) to increase blood pH or 2) retaining CO2 (hypoventilation) to decrease the pH. The
kidney has the ability to 1) excrete H+ and retain HCO3− to increase pH or 2) retain H+ and excrete
HCO3− to lower blood pH. However, renal compensation is slow and occurs over hours to days.
Table 14–1 lists selected respiratory and nonrespiratory disorders associated with hypoxemia,
and Table 14–2 presents selected respiratory and nonrespiratory disorders that can result in acid–
base abnormalities.
ELECTROLYTES AND ANION GAP

Electrolytes are defined as either positively (cations) or negatively (anions) charged ions in the blood.
Four freely circulating electrolytes are typically considered when evaluating acid–base disturbances
(Na+, K+, Cl−, and HCO3−). Blood gas and pH results are most important when investigating acid–
base disruptions, and electrolytes play an important role in identifying the nature of the problem.
Modern blood gas analyzers have expanded test menus to include electrolytes, facilitating their use
in the evaluation of patients with suspected respiratory and/or metabolic disorders.
TABLE 14–1 Selected Disorders Associated With Hypoxemia

Disorder Basis of Abnormality
Chronic bronchitis Impaired ventilation of lung

Emphysema Impaired ventilation of lung
Asthma Impaired ventilation of lung
Pneumoconioses Impaired ventilation of lung
Central or peripheral neuromuscular disorders Impaired ventilation of lung
Right-to-left shunts of great vessels Impaired perfusion of blood into lungs
Pulmonary embolism and pulmonary infarction Impaired perfusion of blood into lungs
Sarcoidosis Impaired diffusion of gases into blood
Selected lung cancers Impaired diffusion of gases into blood

TABLE 14–2 Selected Disorders Associated With Acid–Base Abnormalitiesa

Disorder Metabolic Alterations
Selected causes of metabolic acidosis:

Arterial pH decreased with arterial HCO3− decreased (often from increased acid production associated with
an elevated anion gap or impaired renal elimination) as a primary change and arterial pCO2 decreased as a
compensatory change
Uncontrolled diabetes Increased ketoacids, increased anion gap

Methanol intoxication Increased formic acid, increased anion gap
Tissue hypoxia Increased lactic acid, increased anion gap
Renal failure from a variety of causes Impaired H+ excretion and/or HCO3− absorption
Gastrointestinal HCO3− loss from diarrhea Decrease in HCO3− results in a relative increase in acidity,
normal anion gap, increased Cl−, decreased Na+ and K+
Selected causes of respiratory acidosis:

Arterial pH decreased with arterial pCO2 increased as a primary change and arterial HCO3− increased
as a compensatory change
Neuromuscular disorders that decrease Decreased pCO2 excretion results in increased

breathing such as brain stem injury, carbonic acid (H2CO3)
myasthenia gravis, and poliomyelitis
Severe pulmonary emboli, infections, lung Decreased pCO2 excretion results in increased
cancers, asthma, COPD, and respiratory distress carbonic acid (H2CO3)
syndrome with impaired ventilation, perfusion,
and/or diffusion
Selected causes of metabolic alkalosis:

Arterial pH increased with arterial HCO3− increased as a primary change and arterial pCO2 increased
Vomiting or nasogastric suction Loss of stomach acids (HCl); decreased Cl−;
increased HCO3− reabsorption by the kidneys
Diuretic (ab)use Decreased H+ and K+ from increased renal excretion;
increased HCO3− reabsorption by the kidneys
Cushing syndrome and hyperaldosteronism Decreased K+ from increased renal excretion; leads to
increased H+ excretion and HCO3− reabsorption by the kidneys
Selected causes of respiratory alkalosis:

Arterial pH increased with arterial pCO2 decreased as a primary change and arterial HCO3− decreased
Hyperventilation in response to Increased pCO2 excretion results in decreased
hypoxemia from a variety of causes carbonic acid (H2CO3)
Hyperventilation without hypoxemia as a Increased pCO2 excretion results in decreased
stimulus, as in anxiety and central nervous carbonic acid (H2CO3)
system disorders that increase the respiratory rate
Blood pH = 6.1 + (log[arterial HCO3− concentration]/[0.03 × pCO2]) is the equation that associates the measured
a
parameters in this table.
The anion gap refers to the difference between the major free cations (Na+ and K+) and free The anion gap refers to
anions (Cl− and HCO3−). The calculation of the anion gap from measured electrolyte concentrations the difference between
is critical for evaluation of acidosis. An elevated anion gap occurs when acid anions (such as lactate the major free cations
or ketones) are present. The amount of the increase in anion gap is equal to the amount of acid (Na+ and K+) and free
present because an increase in acid results in a proportional decrease in bicarbonate. Table 14–2 anions (Cl− and HCO3−).
presents selected metabolic acidoses associated with an elevated anion gap. The calculation of
the anion gap from
measured electrolyte
PLEURAL FLUID ANALYSIS concentrations is critical
for evaluation of acidosis.
There are a number of disorders associated with the accumulation of fluid in the pleural space
(Table 14–3). Pleural fluid can be collected by a pleural tap, also known as thoracentesis. An
initial evaluation of color, physical characteristics, and odor of the fluid may help identify the
TABLE 14–3 Selected Disorders With Transudate or Exudate Formation

Basis of Abnormality
Disorders associated with transudate formation
Congestive heart failure An increased systemic venous pressure and pulmonary

capillary pressure, due to the decreased effectiveness
of the heart as a pump, results in leakage of fluid into
the pleural space
Hypoalbuminemia Decreased albumin results in a low vascular

osmotic pressure and fluid accumulation
in body tissues and cavities, including the pleural space
Disorders associated with exudate formation
Pulmonary embolism and pulmonary Blockage of blood vessels results in tissue damage and
infarction leakage of fluid, or in some cases whole blood, into the
pleural space
Pulmonary infections Tissue damage results in leakage of fluid into the

pleural space
Lung tumors Tissue damage results in leakage of fluid into the

pleural space
Autoimmune diseases with pulmonary involvement Tissue damage results in leakage of fluid into the
pleural space
Trauma to the lung or chest wall Tissue damage results in leakage of fluid into the
pleural space
source. Next, the fluid is classified as an exudate or a transudate to limit the differential diagnosis
and help identify the cause of its accumulation. Exudates and transudates are defined by the fol-
lowing criteria:
• Exudate—A filtrate of plasma out of the blood vessel, resulting from capillary damage or
lymphatic obstruction (ie, significant loss of the blood/tissue barrier), with a relatively high
concentration of protein (>3.0 g/dL) and:
Pleural fluid total protein
(a) > 0.5
Serum total protein
(b) Pleural fluid lactate dehydrogenase (LDH) > 20 IU/dL
Pleural fluid LDH
(c) > 0.6
Serum LDH
• Transudate—An ultrafiltrate of plasma with a relatively low protein concentration
(<3.0 g/dL) and values for ([pleural fluid total protein]/[serum total protein]), pleural
fluid LDH, and ([pleural fluid LDH]/[serum LDH]) below what is required to define
the fluid as an exudate.
Pleural fluid total protein
(a) < 0.5
Serum total protein
Pleural fluid LDH

(b) < 0.6
Serum LDH
Other laboratory testing on pleural fluid, while not diagnostic, may provide useful informa-
tion in identifying the source of the fluid accumulation. These include total cell counts and dif-
ferential, Gram stain, pH, glucose, lactate, amylase, triglycerides, and tumor markers. The section
“Infections of the Lung and Pleurae” in Chapter 5 contains information on pleural fluid testing
that is specific for infections.
BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS

Analysis of the components of BAL fluid is an important diagnostic tool in assessment of numer-
ous respiratory disorders. BAL analysis is most helpful when used in conjunction with clinical
data and imaging results to aid in the diagnosis of pulmonary infections, particularly ventilator-
acquired pneumonia, interstitial lung diseases, and lung cancers, and for monitoring of the
allograft postlung transplant. Several aliquots of warmed saline are instilled in different areas of
the lungs. At least 30% of the instilled fluid is carefully aspirated. BAL fluid is collected with the
aid of a bronchoscope.
Bacterial cultures of the pooled fluid sample help identify an infectious cause of respiratory
disease. Analysis of physical characteristics of the BAL collections also aids in the differentiation
of disease. Bloody BAL fluid may indicate a diffuse alveolar hemorrhage, while cloudy BAL fluid
suggests a diagnosis of pulmonary alveolar proteinosis. BAL fluid can also be processed to allow
analysis of soluble biomarkers and cells. Studies of the BAL cell pellet include bacterial cultures,
WBC count and differential, and Gram stain.
LABORATORY TESTS USEFUL IN THE DIAGNOSIS

OR MANAGEMENT OF PULMONARY DISEASES
There are a number of pulmonary diseases for which laboratory tests (other than blood gases,
BAL, and exudate/transudate determination discussed above) are useful in establishing a diag-
nosis. The most common of these are described below, while the rarer disorders are listed in
Table 14–4 with their accompanying clinical laboratory test results. The infectious diseases of
TABLE 14–4 Selected Respiratory Diseases and Laboratory Tests Useful

in Their Diagnosis or Management
Disorder Results/Comment
Sarcoidosis Elevated serum angiotensin-converting enzyme is found in 30%-80%

of sarcoidosis cases and may be a surrogate marker of disease burden; a
CD4/CD8 ratio from BAL fluid >3.5 suggests sarcoidosis
Pulmonary embolism A diagnostic algorithm based on a clinical significance score combined

with radiography and laboratory testing is most accurate in predicting an
embolism. A negative test result for D-dimer (a fibrin degradation product)
in a patient with low-to-moderate clinical probability effectively rules out
PE. A pulmonary embolism is confirmed with a multidetector CT pulmonary
angiogram in patients with a high clinical probability or a positive D-dimer
test; elevated BNP and troponin measurements are associated with a poor
prognosis in patients with PE (see Chapter 8 for a related discussion in the
section “Deep Vein Thrombosis and Pulmonary Embolism”; thrombosis is
also reviewed in Chapter 11)
Alpha-1 antitrypsin deficiency Decreased serum alpha-1 antitrypsin; AAT phenotype assay to identify
protein variants; molecular testing of the SERPINA1 gene to identify allelic
variants associated with reduced activity in adults with early onset COPD
or emphysema. (see Chapter 16 for a discussion of alpha-1 antitrypsin
deficiency)
Goodpasture syndrome Increased concentrations of serum glomerular basement membrane (GBM)

antibodies are found in Goodpasture syndrome; ANCA antibody testing
helps classify disease and rule out other syndromes; blood cell counts
are important to monitor anemia; and renal function tests are useful for
detection of renal failure
Pulmonary vasculidites Discussed in the section “Vasculitis” of Chapter 8 (such as Wegener

granulomatosis and Churg–Strauss syndrome)
Cystic fibrosis Discussed in Chapter 7

Autoimmune-related Discussed in Chapter 3
the respiratory tract and pleurae are presented in Chapter 5. Pulmonary function tests provide
information on airflow and lung volumes. Spirometry assesses pulmonary mechanics by quan-
titating the volume of air moved in different inspiratory and expiratory maneuvers, and the rate
at which the air is moved. In addition, the uptake of a gas can be measured as an indicator of an
impaired alveolar–capillary interface. These tests on airflow and gas exchange often do not iden-
tify a specific respiratory disorder, but they can suggest a category of diseases that may account for
the airflow abnormalities. Because pulmonary function tests are performed outside the clinical
laboratory, they are not discussed further in this text.
Radiologic studies, particularly plain chest radiographs, computed tomographic (CT) scans,
magnetic resonance imaging (MRI), positron emission tomography (PET) scans, and nuclear
medicine studies such as ventilation/perfusion scanning, play a prominent role in the diagnosis
of pulmonary disorders.
COMMON LUNG DISORDERS

Asthma
Asthma is a chronic disease associated with reversible inflammation of the bronchial walls leading
to narrowing of the airways. It is among the most common chronic diseases, and its prevalence
is rising particularly in children. Acute onset of bronchial inflammation can lead to an asthma
attack, with severe forms being life-threatening. While the exact cause of bronchial inflammation
is unknown, there are numerous triggers including: environmental allergens (pollen, dust, and
others), chemical allergens (cleaning agents, smoke, and others), exercise, stress, cold air, and
medications.
Diagnosis and Management

The diagnosis of asthma is challenging because the clinical signs and symptoms overlap with
many other respiratory diseases. Laboratory testing to rule out diseases such as cystic fibrosis (see
Chapter 7) and infections (see Chapter 5) is important for the evaluation. Diagnosis begins with
assessment of airway obstruction with spirometry and chest x-ray. The diagnostic evaluation for
asthma must involve identification of inflammatory triggers. Identification of elevated concentra-
tions of IgE in the plasma indicates a generalized allergic response. Antigen-specific IgE testing
identifies immune responses to specific allergens and is useful in young children and patients
with a contraindication to skin testing.
Initial diagnosis and monitoring of patients with an acute asthmatic reaction involves assess-
ment of oxygenation for disease severity and pulmonary function. Evaluation of arterial blood
gases also assesses oxygenation status. Evaluation of electrolytes, pH, and anion gap is helpful in
evaluating acid–base status, pulmonary function, and tissue hypoxia (Table 14–2).
Chronic Obstructive Pulmonary Disease

Like asthma, COPD is a chronic inflammatory lung disease associated with airway obstruction.
Inflammation leads to thickening of the airway wall and decreased air flow, as well as destruction
of the alveoli and increased airspace. Patients with COPD have 2 main conditions, emphysema
or chronic bronchitis. The biggest risk factor for both is cigarette smoking, followed by exposure
to pollution. Currently, COPD is a major cause of morbidity and mortality worldwide, because it
can lead to respiratory failure.
Diagnosis and Monitoring

A diagnosis of COPD is confirmed by the presence of clinical symptoms such as chronic cough,
wheezing, and/or respiratory failure, combined with airflow obstruction determined by spirom-
etry. Patients with COPD are followed by measuring arterial blood gases to assess oxygen status
and monitor the benefit of long-term oxygen therapy. Exacerbations and increased disease sever-
ity are commonly monitored by measuring a complete blood count (CBC) to assess the level of
inflammation, testing for respiratory infections (Chapter 5), and measurement of electrolytes, pH,
and anion gap to assess acid–base status, pulmonary function, and tissue hypoxia (Table 14–2).
Respiratory Distress Syndrome

Acute Respiratory Distress Syndrome (ARDS)
ARDS is the rapid onset of respiratory failure due to systemic inflammation, trauma, or severe
pulmonary infection in anyone over 1 year of age. It is associated with significant morbidity and
mortality primarily due to oxygen deprivation and multisystem organ failure that results from
pulmonary failure. A consensus group published the Berlin definition of ARDS as hypoxemia
with bilateral lung infiltrates and/or respiratory failure within 1 week of a clinical insult, with
new or worsening respiratory symptoms in the absence of cardiovascular insult or left pulmonary
hypertension.
Diagnosis
The diagnosis of ARDS requires a careful clinical history to identify a recent clinical insult and/or
the timing of new-onset respiratory symptoms. Chest x-ray or CT scan should be performed to
visualize bilateral opacities. Cardiovascular ischemia and fluid overload should be ruled out by
echocardiogram and/or cardiac biomarker analysis. Hypoxemia is classified by measuring arte-
rial blood gasses and calculating the ratio of arterial partial pressure of oxygen to the fraction of
inspired oxygen (Pao2/Fio ). The Berlin definition divides hypoxemia into mild (Pao2/Fio between
2 2
200 and 300 mm Hg), moderate (Pao2/Fio between 100 and 200 mm Hg), and severe (Pao2/Fio
2 2
<100 mm Hg).
Neonatal Respiratory Distress Syndrome

RDS of the newborn is most commonly associated with incomplete development of the fetal
lungs. The pulmonary system is one of the last to completely develop, and as a result, RDS is a
common cause of morbidity and mortality in preterm infants. Symptoms of RDS begin within
a few hours of birth due to a deficiency of pulmonary surfactant. Surfactant, a mixture of phos-
pholipids and proteins, coats the alveolar surfaces and separates alveolar airspace from liquid-
coated lung epithelial cells, preventing lung collapse during exhalation. RDS patients suffer both
lung collapse and hyperextension of alveoli leading to fibrosis, or hyaline membrane disease. The
alveoli in an RDS lung are perfused, but not ventilated, resulting in hypoxia, hypercapnia, and
respiratory acidosis.
RDS can be addressed by preventing preterm births or by administration of corticosteroids Respiratory distress
at least 48 hours prior to a premature birth. Corticosteroids induce surfactant production, sig- syndrome of the newborn
nificantly reducing neonatal morbidity and mortality due to RDS. Assessment of FLM status is is most commonly
essential for clinical decisions for women with symptoms of preterm labor and for those whose associated with
labor is induced prior to 39 weeks gestation. incomplete development
of the fetal lungs. The
pulmonary system is one
Selected Laboratory Tests for Assessment of Fetal Lung Maturity of the last to completely
develop, and as a result,
FLM is most commonly assessed by testing the amount of surfactant in the amniotic fluid in RDS is a common cause of
women after 30 weeks gestation. The diagnostic test most commonly used to assess FLM is the morbidity and mortality
lamellar body count (LBC) assay. Surfactant is packaged into storage granules called lamellar in preterm infants.
bodies that pass into amniotic fluid in the third trimester of pregnancy. LBCs >50,000/μL suggest
maturity. Lamellar bodies are similar in size to platelets and can be counted on a standard whole
blood counter.
Another method to assess fetal lung maturity is through calculation of the surfactant–albumin
(S/A) ratio. The S/A ratio increases throughout gestation proportionally with lung maturity.
Surfactant-based phospholipids, particularly lecithin (phosphatidylcholine) and phosphatidyl-
glycerol (PG), also increase in the amniotic fluid during fetal lung maturation, while lipids not
originating from the lung, such as sphingomyelin, remain fairly constant throughout gestation. Fetal lung maturity can
Because of this, FLM can also be predicted by measuring the lecithin–sphingomyelin ratio. An also be predicted by
L/S ratio >2.0 indicates lung maturity. Qualitative detection of PG in amniotic fluid is a rapid and measuring the lecithin–
sensitive alternative for predicting FLM in late pregnancy. PG measurements are particularly use- sphingomyelin ratio. An
ful in blood and meconium-contaminated amniotic fluid specimens as all other tests described L/S ratio >2.0 indicates
above are affected by these contaminants. lung maturity.
Lung Cancer
Lung cancer, defined as any tumor of the respiratory epithelium or pneumocytes, is the leading
cause of cancer-related mortality worldwide. The leading risk factor for lung cancer is cigarette
smoking, accounting for up to 90% of cases. Exposure to environmental carcinogens, irradiation,
and genetic disorders are also risk factors for developing lung cancer. There are 2 main types of
lung cancers: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC).
Among NSCLCs the most common lung tumor type is adenocarcinoma. Most lung cancers are
caused by acquired mutations, amplifications, or rearrangements in oncogenes including epider-
mal growth factor receptor (EGFR), fibroblast growth factor receptor type 1 (FGFR1), anaplastic
lymphoma kinase (ALK), KRAS, NRAS, BRAF, HER2, PTEN, and MET, among others. Although
some lung cancers are discovered in asymptomatic patients, the most common clinical signs
include coughing up blood, wheezing, and shortness of breath.
Diagnosis and Monitoring

The diagnostic workup for lung cancer begins with discovery of a new pulmonary mass by chest
x-ray, CT scan, or MRI. A definitive diagnosis of lung cancer and type is determined through
histological and immunohistochemical analysis of tumor tissue. Molecular testing of advanced-
stage adenocarcinoma type (NSCLC) is required to direct therapy. In particular, patients with
EGFR mutations are more likely to respond to tyrosine kinase inhibitor (TKI) therapy and have
longer progression-free survival. Patients with a KRAS gene mutation with or without an EGFR
mutation do not respond to TKI therapy and should be treated alternatively. Advanced-stage
non-small cell lung adenocarcinomas should also be tested for rearrangements in the ALK gene.
About 5% to 10% of all NSCLC patients carry this rearrangement, which can be treated with an
ALK inhibitor.
Prior to initiating therapy, baseline CBC and liver function panel should be measured to
screen for metastases. Response to therapy and tumor recurrence can be monitored by perform-
ing regular chest x-rays and/or CT scans, as well as a CBC and liver function tests. Measurement
of cytokeratin 19 fragments (CYFRA 21-1) in serum is useful for assessing prognosis in early and
late stages, and in monitoring therapy in advanced stages of NSCLC. Serum neuron-specific eno-
lase (NSE) may be useful in monitoring therapy and tumor recurrence in both NSCLC and SCLC.
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C H A P T E R
The Gastrointestinal Tract

Michael Laposata and D. Robert Dufour 15
LEARNING OBJECTIVES
1. Understand the relative contributions of clinical laboratory tests and other
diagnostic studies in the evaluation of the patient for a disorder of the
gastrointestinal tract.
2. Learn the appropriate selection of diagnostic tests required to establish
a diagnosis of ulcer disease from Helicobacter pylori infection.
3. Select the most appropriate tests for evaluation of suspected celiac disease,
and learn the situations where results may be misleading.
4. Describe the recommended approaches to screening for colon cancer,
and the benefits and limitations of laboratory tests for this purpose.
CHAPTER OUTLINE
Dyspepsia, Ulcer Disease, and H. pylori 351 Colorectal Cancer 354
Celiac Sprue 353
M
ost diseases of the gastrointestinal tract can be directly visualized by endoscopy or from
a histopathologic review of a biopsy obtained during the endoscopic procedure. In
addition, many gastrointestinal tract disorders can be identified with various imaging
studies. This accessibility of lesions for direct examination and biopsy has limited the need for
clinical laboratory tests in identifying most gastrointestinal disorders. However, because imaging
studies are often expensive, and endoscopic procedures are both expensive and invasive, there
is a clinical need for laboratory tests to aid in the diagnosis and management of persons with a
number of gastrointestinal disorders. Infectious diseases involving the gastrointestinal tract are
numerous, and are discussed in Chapter 5. The clinical laboratory plays an important role in iden-
tifying pathogenic microorganisms of the gastrointestinal tract.
The clinical laboratory also plays a role in the evaluation of dyspepsia (abdominal discomfort
caused by acid), and/or ulcer disease, particularly that induced by Helicobacter pylori infection; in
the recognition and monitoring of celiac sprue; and in the detection and genetic profiling of colon
cancer. Laboratory tests for these disorders are presented in this chapter.
DYSPEPSIA, ULCER DISEASE, AND H. PYLORI

Description
According to the American Gastroenterological Association (AGA), dyspepsia is defined as
chronic or recurrent pain or discomfort centered in the upper abdomen. Other conditions (par-
ticularly reflux of acid into the esophagus, referred to as gastroesophageal reflux disease [GERD])
can also cause abdominal discomfort, and it is often difficult to specifically characterize the
type and location of discomfort. About 10% of those in the United States with upper abdominal
351
352 CHAPTER 15 The Gastrointestinal Tract
symptoms are found to have peptic ulcers, with a wide range between different countries. Other
causes include gastritis related to use of nonsteroidal anti-inflammatory agents, and “functional
dyspepsia,” in which no obvious pathology is present in the stomach.
The major cause of peptic The major cause of peptic ulcer disease is infection with H. pylori. The infection is most
ulcer disease is infection likely to occur in childhood, especially if the children are living in low socioeconomic conditions.
with H. pylori. Not all In developed countries, H. pylori infection prevalence increases with age. Not all patients with
patients with H. pylori H. pylori infection develop ulcer disease, as many suffer from dyspepsia without ulcers. The infec-
infection develop ulcer tion initially produces an acute gastritis that lasts 1 to 4 weeks. Once infected, however, chronic
disease, as many suffer active gastritis occurs in the majority of individuals and may lead to more serious outcomes. Espe-
from dyspepsia without cially when infected in early childhood, individuals are at risk for the development of multifocal
ulcers. atrophic gastritis and over time, subsequently, have an increased risk for gastric adenocarcinoma.
Diagnosis
The evaluation of individuals with dyspepsia depends on age and the severity of symptoms.
According to guidelines, direct visualization of the upper gastrointestinal tract by endoscopy is
the preferred initial step in persons over age 55 years, or in younger patients who have a fam-
ily history of gastric cancer or who also have more worrisome symptoms such as weight loss,
difficulty swallowing food, recurrent vomiting, or gastrointestinal bleeding. In younger patients
without these symptoms, the recommended approach is to test for the presence of active H. pylori
infection and treat infected individuals. Those without evidence of infection are treated with
drugs that inhibit acid production. Persons who do not respond to these treatments should then
have an endoscopy.
Laboratory tests for H. pylori can be separated into those that identify exposure and those
that detect active infection (Table 15–1). Because H. pylori is able to split urea (it is a urease-
positive organism), its presence can be detected by ability of the individual to metabolize urea. The
urea breath test (UBT) involves ingestion of a food product containing urea labeled with a small
amount of radioactive carbon. If urease activity is present, the urea will be split into ammonia and
carbon dioxide. The amount of radioactive carbon dioxide in the breath correlates with urease
activity. The UBT has a high accuracy, and its performance is simple. Use of drugs that suppress
acid production may cause falsely negative UBTs.
H. pylori has a unique surface antigen that can be detected in the stool of infected individuals,
but not in those with inactive infection. Stool antigen testing with kits using monoclonal antibod-
ies to the antigen is sensitive and specific. These kits have a high accuracy, for both initial diagno-
sis of H. pylori and posttreatment follow-up testing. Both the UBT and the stool antigen test can
be used to evaluate the success of antibiotic treatment for H. pylori. Successful treatment should
lead to loss of stool antigen (after several weeks) and the loss of urease activity in the stomach.
Serologic tests for IgG antibodies to H. pylori indicate past or current infection. They have
a sensitivity and a specificity of only about 85% to 90%, and have largely been replaced by tests
TABLE 15–1 Diagnosis of H. pylori Infection—Selected Diagnostic Recommendations

From the Maastricht IV/Florence Consensus Meeting in 2010
The diagnostic accuracy of a stool H. pylori antigen test is equivalent to the urea breath test if a validated
laboratory-based stool antigen test involving a monoclonal antibody is used
Because of the variability in the accuracy of different commercial tests, only validated IgG serology tests for
H. pylori infection should be used
If there has been recent use of antimicrobial or antisecretory drugs, or ulcer bleeding, atrophy, or gastric
malignancies, a validated IgG serology test for H. pylori may be used to assess current or past infection
In patients treated with proton pump inhibitors, these drugs should be stopped, if possible, for 2 weeks before
testing by culture, histology, and/or rapid urease test using biopsy material; or by the urea breath test or stool
H. pylori antigen test. If proton pump inhibitors cannot be discontinued, a validated IgG serologic test for
H. pylori can be performed
In a region or population of patients in which resistance to clarithromycin is high, culture and standard
susceptibility testing to clarithromycin and other antimicrobial agents should be performed to determine if
these agents will be clinically effective
CHAPTER 15 The Gastrointestinal Tract 353
that directly identify the organism. Serology is the only test not affected by reduction of bacterial
load in the stomach, and, therefore, serologic testing for H. pylori is not subject to false-negative
results when bacterial load reduction occurs. Unlike the stool antigen and urease tests, serologic
tests remain positive for years after successful treatment, and because of that they are of no use to
monitor the effects of treatment.
If endoscopy is performed, tests involving use of the biopsy material include a rapid urease
test, histology, and culture with susceptibility testing. Susceptibility testing is increasingly impor-
tant to detect resistance to clarithromycin because in resistant patients, the treatment success
with clarithromycin is low. Molecular tests for H. pylori can also be used to detect the organism
in gastric biopsies.
CELIAC SPRUE
Description
Celiac disease is a systemic immune-mediated disorder. In genetically susceptible individuals, it is Celiac disease is an
triggered by dietary gluten. Gluten is a complex of proteins that are found in wheat, rye, and barley. immunologic disorder
Gliadin is the water-soluble component in gluten. Celiac disease is extremely common, affecting caused by immune
0.6% to 1% of the worldwide population. However, only a small percentage of cases of celiac reactions triggered
disease are recognized. Its prevalence is higher in women, with increased incidence in individuals by gluten and related
proteins that are
with an affected first-degree relative, or a relative with type 1 diabetes, Hashimoto thyroiditis, or
components of wheat,
other autoimmune disease. Importantly, genetic background greatly influences the predisposition
barley, and rye grain
to celiac disease. In 90% of patients with celiac disease, the HLA-DQ2 haplotype is expressed
products.
(which is present in only approximately one third of the general population). The HLA-DQ8
haplotype is expressed in 5% of the patients with celiac disease. This genetic predisposition
occurs because the HLA-DQ2 and HLA-DQ8 haplotypes are expressed on the surface of antigen-
presenting cells that bind activated (deamidated) gluten peptides. The HLA-DQ2 and HLA-DQ8
haplotypes are necessary, but their presence alone is not sufficient for the development of celiac
disease. There are dozens of non-HLA genes that confer predisposition to celiac disease, and most
of these are involved in the inflammatory and immune response.
Patients with celiac disease have chronic inflammation of the proximal small intestinal
mucosa. The inflammation can heal when foods containing gluten are excluded from the diet.
The inflammation returns if foods containing gluten are reintroduced. Gluten-associated storage
proteins derived from wheat, barley, and rye undergo partial digestion in the upper gastrointesti-
nal tract. The partial digestion results in the generation of derivatives of the native peptides, and
these specific peptides can elicit an immune response. The enzyme transglutaminase deamidates
glutamine to negatively charged glutamic acid residues in gliadin peptides, which then stimulate
the immune response and the subsequent intestinal injury.
Mildly affected individuals may have symptoms such as bloating, irregular bowel movements,
and cramps (often referred to as irritable bowel syndrome). Celiac disease patients may present
with malabsorption of certain essential nutrients, including iron. About 5% of iron deficiency in
adults is thought to be due to celiac disease. Malabsorption of folate and vitamin D, which may
present clinically as osteoporosis, can also occur.
Diagnosis
Laboratory testing for celiac disease is summarized in Table 15–2.
Serologic Tests
A serologic test for IgA anti-tissue transglutaminase (tTG) antibodies is recommended as the
initial testing for individuals who do not have a concomitant IgA deficiency. This is the most
widely used test and has a sensitivity and a specificity over 98%, especially now that human tTG
is used in the test as a reagent. As many as 3% of those with classic celiac disease have a deficiency
of IgA, which results in a falsely negative test result. In a person with a high suspicion for celiac
disease and a negative anti-tTG result, measurement of IgA to determine if the patient is IgA
deficient is recommended. In persons with IgA deficiency, IgG, instead of IgA, anti-tTG antibodies
can be measured. Another alternative for IgA-deficient patients is the measurement of the IgG
TABLE 15–2 Commonly Used Diagnostic Tests for Celiac Disease

Test Advantages Disadvantages
Tissue transglutaminase Most reliable noninvasive test Falsely negative with IgA deficiency
(tTG) IgA antibodies and first-level screening test (3% of patients with celiac disease)
High sensitivity and specificity May be negative if on low-gluten diet
Tissue transglutaminase Useful in patients with Widely variable sensitivity
(tTG) IgG antibodies IgA deficiency and specificity
IgA antiendomysial antibodies May be useful in patients Sensitivity for celiac disease less
with borderline results for tTG than IgA anti-transglutaminase
antibodies antibody test
IgG deamidated gliadin Useful in patients with IgA Not as sensitive or specific as
peptide antibodies deficiency and in young children tTG IgA antibodies
HLA-DQ2 or HLA-DQ8 High negative predictive value for Test is complex and expensive
celiac disease
Small bowel biopsy Reliable test, considered Requires endoscopy and biopsy
gold standard Very expensive
Reflects response to treatment
deamidated gliadin peptide antibodies. Tests for antibodies to deamidated gliadin peptides are
less sensitive and less specific in adults for diagnosis of celiac disease, but they are more sensitive
than anti-tTG assays in children. Antibody tests to gliadin are less likely to be positive in milder
cases, and, like the other serologic tests, often become negative when gluten is eliminated from
the diet. Some patients may be monitored with antibody levels to gliadin to monitor compliance
with treatment.
The endomysium is a connective tissue that ensheaths each individual muscle fiber. Antien-
domysial antibodies are present in patients with celiac disease. They are useful in the diagnosis
of the disease, but do not cause any direct symptoms associated with muscles. The presence of
antiendomysial antibodies is nearly 100% specific for active celiac disease, but these antibodies
are found in other autoimmune diseases, and for that reason this antibody measurement should
be used as a confirmatory test for borderline cases initially tested with an anti-tTG antibody assay.
Patients with celiac disease can be differentiated from patients with simple gluten sensitiv-
ity and patients with wheat allergy because the antibodies found in celiac disease are absent in
those with gluten sensitivity or wheat allergy. In addition, for celiac disease, the interval between
exposure to gluten and onset of symptoms is weeks to years. This is in contrast to simple gluten
sensitivity where the interval between exposure and onset of symptoms is hours to days, and
wheat allergy where the interval is minutes to hours.
Biopsy
The diagnosis of celiac sprue currently requires endoscopy with biopsy of the duodenum. In
severe cases, there is atrophy of villi and flattening of the mucosa, but milder cases may show only
lymphocytes infiltrating the mucosa. In children, recent guidelines suggest that a biopsy may not
be required if there are typical symptoms and a high titer of anti-tTG antibodies, along with HLA-
DQ2 and/or HLA-DQ8 genotypes.
COLORECTAL CANCER
Description
Because colorectal cancer is the second most common cause of cancer death in both men and
women, and because early detection is associated with better survival, there is widespread accep-
tance of the benefits of screening those over age 50 years for colorectal cancer (see Table 15–3).
Screening may also detect adenomas of the colon, which have been recognized to be the
precursors of most cases of invasive cancer. The most sensitive screening test for colorectal can-
cer is colonoscopy.
CHAPTER 15 The Gastrointestinal Tract 355
TABLE 15–3 Screening Test Guidelines for Colorectal Cancer for Average-risk
Individuals by the US Preventive Services Task Force, American
Cancer Society, Multi-society Task Force on Colorectal Cancer, and
American College of Radiology
Test Frequency of Testing Comments
Stool-based tests
Sensitive guaiac fecal occult Annual Poor sensitivity for detecting colorectal carcinoma
blood test (FOBT) (less than 50%)
Or
Fecal immunochemical Annual Sensitivity for detecting colorectal cancer
test (FIT) 60%-85%, but different commercial products
with differing sensitivity and specificity
Structural examinations of the colon

Colonoscopy Every 10 years Structural examination of the colon preferred
beginning at age 50, individualized between age
75 and 84, and not recommended after age 85
Diagnosis—Screening for Colorectal Cancer

Because colonoscopy is an invasive and expensive procedure with risks involved, other approaches
to screening are also approved. These include fecal occult blood testing (FOBT) and fecal immu-
nochemical testing (FIT). Both of these tests involve testing stool for the presence of blood. The
FOBT uses guaiac, a reagent that reacts with hemoglobin and a number of other substances to
produce a blue color when blood is present. Guaiac tests react with animal hemoglobin as well,
so restrictions on meat intake are required for several days before samples are collected. Iron and
certain plants also can cause a blue color with guaiac, and their intake should also be restricted.
The testing is done by having an individual take 2 samples from 3 consecutive bowel movements,
and smearing these small samples onto cards that are sent to a site where they can be tested. This
test has a poor sensitivity for detecting colorectal cancer.
The fecal immunochemical test (FIT), using antibodies to human hemoglobin, is slightly
more expensive, but does not require dietary restriction before testing. Both types of tests for
hemoglobin are approved in commonly used guidelines. However, the FIT has a much higher
sensitivity than the FOBT for detection of colorectal cancer and also for detecting advanced
adenomas that can develop into cancer.
Colonoscopy needs to be repeated infrequently, every 10 years if no adenomas are found Colonoscopy needs to be
starting at age 50 in persons who are not considered to be at high risk for colorectal cancer. repeated infrequently,
Colonoscopy should be more frequently performed for those with adenomas or with a strong every 10 years if no
family history of colon cancer. FIT or FOBT should be performed every year. Any abnormal adenomas are found,
results should be followed up by colonoscopy. although more frequently
Colorectal cancer, like most cancers, is associated with a number of mutations in genes in those with adenomas
related to cell growth and regulation. It is possible to identify DNA from shed cells in stool. Some or with a strong family
history of colon cancer.
studies have shown that testing for multiple mutations in genes associated with colon cancer has a
higher sensitivity than FOBT, although not as high as colonoscopy. The DNA test requires special
handling, with collection of stool into a container that has been stored frozen, and rapid delivery
after collection to the testing laboratory. It is similar in expense to colonoscopy at present. Labora-
tory testing to screen patients for colorectal cancer is summarized in Table 15–3.
Diagnosis—Genetic Profiling for Colorectal Cancer

Histologically, the most common subtype of colorectal cancer is adenocarcinoma. Colorectal ade-
nocarcinomas can arise upon acquisition of a variety of mutations over many years. These genetic
alterations can lead to the conversion of normal colonic epithelium first to adenoma, and then
to carcinoma, and this carcinoma frequently metastasizes. There has been increasing recognition
that some of the genetic alterations can be used as prognostic markers for outcome and can be
useful in the selection of specific therapies for the patient with colorectal cancer. As an example,
1 genetic profile for colon cancer involves the analysis of 63 mutations in the following 7 genes—
KRAS, BRAF, PIK3CA, AKT1, SMAD4, PTEN, and NRAS.
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Malfertheiner P, et al. Management of Helicobacter pylori infection—the Maastricht IV/Florence consensus
report. Gut. 2012;61:646–664.
McCallion WA, et al. Helicobacter pylori infection in children: relation with current household living
conditions. Gut. 1996;39:18.
NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. Helicobacter pylori in
peptic ulcer disease. JAMA. 1994;272:65.
Parsonnet J. Helicobacter pylori in the stomach: a paradox unmasked. N Engl J Med. 1996;335:278.
Talley NJ; American Gastroenterological Association. American Gastroenterological Association medical
position statement: evaluation of dyspepsia. Gastroenterology. 2005;129:1753–1755.
Celiac Sprue:
Agardh D. Antibodies against synthetic deamidated gliadin peptides and tissue transglutaminase for the
identification of childhood celiac disease. Clin Gastroenterol Hepatol. 2007;5:1276–1281.
Boettcher E, Crowe SE. Dietary proteins and functional gastrointestinal disorders. Am J Gastroenterol.
2013;108:728–736.
Fasano A, Catassi C. Celiac disease. N Engl J Med. 2012;367:2419–2426.
Rostom A, et al. The diagnostic accuracy of serologic tests for celiac disease: a systematic review.
Gastroenterology. 2005;128:S38–S46.
Rostom A, et al. American Gastroenterological Association (AGA) Institute technical review on the
diagnosis and management of celiac disease. Gastroenterology. 2006;131:1981–2002.
Colon Cancer:
Ahlquist DA, et al. Next-generation stool DNA test accurately detects colorectal cancer and large
adenomas. Gastroenterology. 2012;142:248–256.
Levin B, et al. Screening and surveillance for early detection of colorectal cancer and adenomatous
polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force
on Colorectal Cancer, and the American College of Radiology. Gastroenterology. 2008;134:1570–1595.
Lieberman D. Colorectal cancer screening: practice guidelines. Dig Dis. 2012;30(suppl 2):34–38.
Schoen RE, et al. Colorectal-cancer incidence and mortality with screening flexible sigmoidoscopy. N Engl
J Med. 2012;366:2345–2357.
Sinatra MA, et al. Interference of plant peroxidases with guaiac-based fecal occult blood tests is avoidable.
Clin Chem. 1999;45:125–126.
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recommendation statement. Ann Intern Med. 2008;149:627–637.
C H A P T E R
The Liver and Biliary Tract

William E. Winter 16
LEARNING OBJECTIVES
1. Identify the laboratory tests useful in the evaluation of liver function, and the
pathophysiology that results in the generation of these abnormal test results.
2. Understand the clinical laboratory evaluation of the patient for viral hepatitis.
3. Associate specific disorders of the liver with the laboratory test results
expected for those clinical diagnoses.
CHAPTER OUTLINE
Introduction 357 Selected Liver Diseases and Laboratory Tests
Plasma Membrane Integrity and Used in the Evaluation of Liver Function 366
Disorders Predominantly Associated Alpha-1 Antitrypsin Deficiency 366
With Elevated Concentrations of Glycogen Storage Diseases 366
Liver-derived Enzymes in the Hemochromatosis 367
Blood 358 Wilson Disease 367
Detoxifying and Excretory Functions Hepatocellular Carcinoma and
of the Hepatobiliary System and Alpha-fetoprotein (AFP) 367
Disorders Associated Predominantly Hepatic Encephalopathy and Ammonia 368
With an Elevated Bilirubin Cholestasis of Pregnancy and Serum
Concentration 361 Bile Acid 368
Synthetic Function of the Liver and Acute (Fulminant) Hepatic Failure 368
Disorders Associated With Low Cirrhosis 369
Circulating Concentrations of Albumin, Primary Biliary Cirrhosis 369
Transthyretin, Retinol-binding Protein, Primary Sclerosing Cholangitis 369
and Coagulation Factors 364 Approach to the Patient With Liver
The Diagnosis of Viral Hepatitis 365 Disease 369
INTRODUCTION
Laboratory evaluation of the hepatobiliary system centers on measurements of: 1) hepatocyte
plasma membrane integrity, 2) measurements of the detoxifying and excretory functions of the
hepatobiliary system, and 3) measurements of the synthetic capacity of hepatocytes.
357
358 CHAPTER 16 The Liver and Biliary Tract
PLASMA MEMBRANE INTEGRITY AND DISORDERS

PREDOMINANTLY ASSOCIATED WITH ELEVATED
CONCENTRATIONS OF LIVER-DERIVED ENZYMES
IN THE BLOOD
With hepatocyte or biliary tract disease, many cellular enzymes are released that enter the circu-
lation. Enzymes indicative of hepatocyte disease are alanine aminotransferase (ALT) and aspar-
tate aminotransferase (AST). Alkaline phosphatase (ALP) elevations relate to biliary tract disease
(Table 16–1).
Enzyme concentrations are usually measured by determining the enzyme activity in serum
or plasma. Such measurements are reported as units per liter or international units per liter, where
the unit is an activity measurement (eg, the rate of appearance of product or disappearance of
substrate per unit time).
Enzymes indicative of Normally, the healthy plasma membrane and various organelles contain (eg, “hold”) enzymes
hepatocyte disease are within the cell. An elevated enzyme level in the blood suggests organ dysfunction because of a
alanine aminotransferase functional or anatomic disruption in the plasma membrane. One way to assess the degree of
(ALT) and aspartate elevation of an enzyme is to calculate the ratio of the patient’s enzyme concentration relative to
aminotransferase (AST). the upper limit of the reference interval. For example, if the upper limit of the reference interval
Alkaline phosphatase for ALT were 40 U/L and the patient’s ALT was 120 U/L, the patient’s ALT would be said to be
(ALP) elevations relate to “3 times above the upper limit of normal.”
biliary tract disease. While not specific for hepatocytes, elevations of ALT and AST are characteristic of hepato-
cellular disease. The major sources of ALT include the liver and the kidney. Lesser amounts are
released from skeletal and cardiac muscle. AST is also found in these organs. ALT is exclusively
localized in the cell cytoplasm. AST is located in the cytoplasm and mitochondria. However, AST
derived from the cytoplasm and mitochondria cannot be distinguished through clinical labora-
tory testing. ALT is more specific for the liver than AST. Usually ALT and AST rise in tandem
in liver disease states. Although there is more AST in hepatocytes than ALT, ALT is metabolized
more slowly than AST accounting for similar concentrations of these enzymes in the patient’s
plasma as released from the liver.
One condition where AST is often elevated to a greater extent than ALT is in chronic liver
disease resulting from chronic alcohol abuse. People with alcoholism are not uncommonly pyri-
doxine deficient because of deficient dietary intake of this vitamin. While both AST and ALT are
pyridoxine dependent for their biochemical activity, ALT is more dependent on pyridoxine than
AST. Thus, a rise in the measured ALT may not be as great as the rise in measured AST because
ALT activity suffers more from pyridoxine deficiency than does AST. If the AST to ALT ratio is
greater than 2 in the setting of chronic liver disease, alcoholic liver disease is strongly suggested.
With cirrhosis of any etiology, enzyme elevations may be modest, or their concentrations may be
surprisingly normal, reflecting a marked loss in hepatocyte mass and, thereby, a loss of enzymes
within the liver.
TABLE 16–1 Enzymes Indicative of Liver Plasma Membrane Integrity

Indicative of hepatocellular disease
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Lactate dehydrogenase (LD)
Indicative of biliary tract disease
Alkaline phosphatase (ALP)
Gamma-glutamyltransferase (GGT)
5′-Nucleotidase (5′-NT)
CHAPTER 16 The Liver and Biliary Tract 359
TABLE 16–2 Lactate Dehydrogenase (LD) Isoenzymes: Subunit Composition

and Distribution
Isoenzyme Subunits Distribution
LD1 H4 Heart, red blood cell, renal cortex
LD2 H3M Heart, red blood cell, renal cortex
LD3 H2M2 Pancreas, lung, lymphocyte, platelet
LD4 HM3 No specific distribution

LD5 M4 Hepatocyte, skeletal muscle, prostate
H, the heart subunit; M, the muscle subunit.
In the past, lactate dehydrogenase (LD) was also regularly employed as a marker of hepa-
tocellular disease. (Note: The older abbreviation for lactate dehydrogenase was “LDH.”) How-
ever, LD is not favored for routine evaluation of the hepatocyte integrity currently because LD
is released with injury of many different tissues. Both ALT and AST are more specific for liver
disease or injury than LD.
Measurement of LD isoenzymes is possible, but there are more informative tests that can be
ordered to evaluate specific organ dysfunction. LD is composed of 4 subunits. The subunits are H
(for heart) and M (for muscle). If required, LD isoenzymes can be determined by electrophoresis.
The subunit composition and major sources of each of the 5 isoenzymes are listed in Table 16–2.
The LD4 isoenzyme provides no specific clinically useful information.
If the total LD is increased in a patient with suspected liver disease, and the patient lacks
skeletal muscle and prostate disease, it is expected that LD5 will be elevated because of the liver
disease. The enzyme marker of choice for the evaluation of skeletal muscle injury or disease is
creatine kinase (CK). If the CK is normal in the setting of an elevated LD5, skeletal muscle is not
likely to be the source of the elevated LD5.
Biliary tract disease produces relatively greater increases in ALP than increases in ALT, AST,
or LD. ALP is associated with the plasma membrane of hepatocytes adjacent to the biliary canalic-
ulus. Obstruction or inflammation of the biliary tract results in an increased concentration of the
ALP in the circulation. Similar to ALT and AST, ALP is not specific for biliary tract disease. ALP
is released by osteoblasts, the ileum, and the placenta. ALP is elevated: 1) in children 2- to 3-fold
over adults because the child’s skeleton is growing, 2) with bone disease involving osteoblasts (eg,
metastatic cancer or following a fracture), 3) in hyperparathyroidism where parathyroid hormone
stimulates osteoblasts through a series of steps that enhances bone resorption (eg, parathyroid
adenoma, hyperplasia, or secondary hyperparathyroidism from vitamin D deficiency or renal Biliary tract disease
disease), 4) in cases of ileal disease, and 5) during the third trimester of pregnancy because the produces relatively
placental isoenzyme is elevated. greater increases in ALP
When the etiology of the elevated ALP is unclear, in the past ALP isoenzymes were deter- than increases in ALT, AST,
mined. However, there are many technical problems with these assays. Today it has proven more or LD. ALP is associated
pragmatic to measure other biliary tract enzyme markers such as gamma-glutamyl transpeptidase with the plasma
(GGT; aka gamma-glutamyltransferase) or 5′-nucleotidase (5′-NT). The proximal convoluted membrane of hepatocytes
tubule of the kidney, the liver, the pancreas, and the intestine are sources of GGT, in decreasing adjacent to the biliary
order of tissue concentration. Within the cell. GGT is located in microsomes and along the bili- canaliculus.
ary tract plasma membrane, GGT is more commonly measured than 5′-NT because GGT test-
ing is widely available on a variety of laboratory instruments. GGT is typically not elevated with
bone disease. Combined elevations of ALP and GGT are compatible with biliary tract disease.
However, if the ALP is elevated to a far greater extent than the GGT (or the GGT is normal), ALP
sources other than the biliary tract, such as bone, must be investigated. GGT elevations occur in
response to alcohol use and anticonvulsants, as GGT is induced by such agents. While there is no
specific biochemical test to prove that a patient suffers from alcohol abuse, carbohydrate-deficient
transferrin levels can be elevated in patients suffering from alcoholism.
Using the information presented, one can interpret of liver enzyme elevations in patients
with suspected liver disease. If the relative increase in ALT or AST over the upper limit of
TABLE 16–3 Causes of Hepatocellular Diseasea

Acute: present for less than 6 months
Common
Viral hepatitis (hepatitis A, B, or C)
Alcoholic hepatitis
Toxic injury
Ischemic injury
Less common
Viral hepatitis that is not hepatitis A, B, or C (includes hepatitis D, hepatitis E,
cytomegalovirus [CMV], Epstein–Barr virus [EBV], and herpes virus)
Autoimmune hepatitis
Wilson disease
Liver disease of pregnancy
Chronic: present for more than 6 months
Viral hepatitis B or C
Drug toxicity
Alcoholic liver disease
Nonalcoholic fatty liver (NAFL)
Inborn errors (include hemochromatosis, alpha-1 antitrypsin deficiency, Wilson disease,
glycogen storage disease, and Gaucher disease)
Autoimmune hepatitis
a
The relative elevations in ALT and AST exceed the relative elevation in ALP.
normal exceeds the relative increase in ALP over the upper limit of normal, the liver disease is
predominantly hepatocellular as opposed to biliary tract.
Causes of acute hepatocellular disease include (Table 16–3) viral hepatitis (eg, hepatitis A,
B, or C), alcoholic hepatitis, toxic injury (eg, acetaminophen poisoning), and ischemic injury (eg,
hypotension). In cases of ischemic injury or toxic injury following an acute toxic ingestion, the
ALT and AST levels can rise and peak within 24 hours of the precipitating event. Less common
causes of acute liver disease include hepatitis due to hepatitis D, hepatitis E, cytomegalovirus
(CMV), Epstein–Barr virus (EBV), and herpes virus; autoimmune hepatitis (marked by posi-
tivity for antinuclear antibodies [ANA], smooth muscle autoantibodies [ASMA], and/or liver–
kidney microsome autoantibodies [anti-LKM1 autoantibodies] and negative antimitochondrial
autoantibodies [AMA]); Wilson disease; and liver disease of pregnancy. Three forms of liver
disease in pregnancy include fatty liver, intrahepatic cholestasis, and hepatic dysfunction associ-
ated with toxemia (eg, part of the HELLP syndrome: hemolysis, elevated LFTs [eg, enzymes], and
low platelets).
Chronic hepatocellular disease is diagnosed when liver disease is present for more than
6 months (Table 16–3). Causes of chronic hepatocellular disease include hepatitis B or C, drug
toxicity (eg, statins, sulfonamides, or INH), alcoholic liver disease, nonalcoholic fatty liver
(NAFL), inborn errors of metabolism, and autoimmune hepatitis. NAFL is one of the most
common causes of nonviral and nonalcoholic liver disease. It can progress to nonalcoholic
steatohepatitis (NASH), cirrhosis, liver failure, and even hepatocellular carcinoma in some cases.
Inborn errors causing chronic liver disease encompass hemochromatosis, alpha-1 antitrypsin
deficiency, Wilson disease, glycogen storage disease (GSD), and Gaucher disease.
Bilirubin is predominantly The AST to ALT ratio can be used to suggest alcoholic liver disease. One can argue that
derived from hemoglobin excluding the setting of alcoholism, hepatocellular disease can be adequately assessed with the
in the normal turnover measurement of ALT alone. However, it is common medical practice to measure both enzymes,
of red blood cells, and and the enzyme measurements are rapidly available and can be performed at low cost in modern
to a lesser extent, from automated laboratories.
myoglobin in muscle. If the relative increase in ALP over the upper limit of normal exceeds the relative increase
in ALT or AST over the upper limit of normal, the liver disease predominantly involves the
biliary tract (Table 16–4). A major manifestation of obstructive biliary tract disease is an
elevated bilirubin concentration. Clinical jaundice results when the total bilirubin exceeds
2 to 3 mg/dL.
TABLE 16–4 Causes of Biliary Tract Diseasea

Failure of formation of the bile ducts
Biliary atresia
Obstruction or obliteration of the bile ducts
Cholelithiasis
Cholangitis
Primary biliary cirrhosis
Primary sclerosing cholangitis
Postsurgical strictures
Parasitic infection
Compression of the bile ducts outside the liver
Pancreatic cancer
Pancreatitis
Hepatic cancers
a
The relative elevation in ALP exceeds the relative elevations in ALT and AST.
DETOXIFYING AND EXCRETORY FUNCTIONS OF THE

HEPATOBILIARY SYSTEM AND DISORDERS ASSOCIATED
PREDOMINANTLY WITH AN ELEVATED BILIRUBIN
CONCENTRATION
A major biochemical responsibility of the liver is to metabolize toxins, drugs, and biologic end
products and excrete many of the resulting metabolites into the bile. The easiest endogenous
end product to assess is the bilirubin concentration in the plasma. Bilirubin is predominantly
derived from hemoglobin in the normal turnover of red blood cells, and to a lesser extent, from
myoglobin in muscle. Red blood cells normally circulate for approximately 120 days. Red blood
cell senescence and destruction in monocytes/macrophages, primarily in the spleen, releases
hemoglobin from red blood cells. Within the phagocyte, hemoglobin is then metabolized to bili-
verdin and finally to bilirubin. The bilirubin then enters the circulation. This form of bilirubin
(ie, “unconjugated” bilirubin) is relatively insoluble in water and is transported to the hepatocyte
bound to albumin. It is not excreted in the urine. Unconjugated bilirubin is normally taken up
into hepatocytes via a transport system. Inside the hepatocyte via the action of UDP-glucuronyl
transferase, either 1 or 2 glucuronide molecules are conjugated to bilirubin, making the biliru-
bin water soluble. Conjugated bilirubin is bilirubin monoglucuronide or bilirubin diglucuronide.
Conjugated bilirubin is then transported across the plasma membrane into the bile canaliculi
along with bile via multiple drug resistance (MDR) transporter proteins. If the concentration of
either conjugated or unconjugated bilirubin rises pathologically, the skin and sclera can develop If the concentration of
a yellowish color, termed jaundice. With marked elevations in bilirubin, patients may acquire a either conjugated or
green hue. Pathologic elevations in water-soluble bilirubin (eg, conjugated bilirubin) can lead to unconjugated bilirubin
bilirubin excretion in the urine (bilirubinuria), causing the urine to develop a yellow-brown or rises pathologically,
green-brown color. the skin and sclera can
Bilirubin is most often measured by reacting the patient’s serum or plasma with Ehrlich develop a yellowish color,
reagent that includes a diazo compound. The conjugated fraction reacts most rapidly with the termed jaundice.
reagent because the conjugated fraction is water soluble. This is termed “direct acting,” or more
commonly, “direct” bilirubin. To measure total bilirubin, solubilizing agents must be added to
the serum or plasma to enhance the reaction of the water-insoluble bilirubin (ie, unconjugated
bilirubin) with the reagents. Caffeine or benzoate can be used for this purpose. Because only
direct and total bilirubin can be measured, indirect (unconjugated bilirubin) is calculated as the
difference between the total and the direct bilirubin. While the terms “direct” and “conjugated”
are used synonymously just as the terms “indirect” and “unconjugated” bilirubin are used synony-
mously, it should be noted that these are approximations. In fact, direct bilirubin measures 70%
to 90% of the conjugated bilirubin, delta bilirubin (biliprotein, see below), and 5% to 10% of the
unconjugated bilirubin.
While the chemical measurement of bilirubin using Ehrlich reagent is the scheme used in
the majority of automated chemistry analyzers, it is necessary to review how bilirubin is mea-
sured using dry slide technology that was originally developed by Kodak. The unique feature of
dry slide technology (as provided in the Vitros series of analyzers) is the ability to spectrophoto-
metrically determine the unconjugated (BU: bilirubin unconjugated) and conjugated bilirubin
(BC: bilirubin conjugated) fractions. The difference between the sum of the BU and BC and the
total bilirubin measured via the Ehrlich reaction is the delta bilirubin (aka biliprotein). Delta
bilirubin is bilirubin that is covalently bound to albumin. Elevated levels of delta bilirubin are
consistent with chronic elevations in bilirubin. However, only 1 analytical system is able to esti-
mate delta bilirubin (ie, dry slide technology) and the calculation of the delta bilirubin has not
yet been shown to be clinically informative.
In most cases involving hepatocellular dysfunction (notably, acute viral hepatitis), the major
relative increase in bilirubin is an increased conjugated bilirubin fraction because transport of
conjugated bilirubin into the bile canaliculus is typically the rate-limiting step in bilirubin excre-
tion. With the failure of transport of conjugated bilirubin into the bile canaliculus, the conjugated
It is useful to classify bilirubin refluxes into the systemic circulation. However, with severe hepatocellular dysfunction,
hyperbilirubinemia as might occur in cases of end-stage liver disease, there can be defective conjugation in addition
as predominantly to defective canalicular transport.
unconjugated or It is useful to classify hyperbilirubinemia as predominantly unconjugated or conjugated. This
conjugated. When the assists in the development of an appropriate differential diagnosis. If the ratio of the conjugated
ratio of conjugated to bilirubin to total bilirubin is less than 0.4, an unconjugated hyperbilirubinemia is present. When
total bilirubin is 0.4 or the ratio of conjugated to total bilirubin is 0.4 or greater, predominantly a conjugated hyperbili-
greater, predominantly rubinemia is present. In neonates, the cutoff ratio is near 0.2 because unconjugated bilirubin is
a conjugated normally much higher in neonates than in children or adults.
hyperbilirubinemia is Causes of unconjugated hyperbilirubinemia involve 3 basic mechanisms: 1) increased red
present. blood cell destruction (“prehepatic”), 2) defects in the transport of unconjugated bilirubin into
the hepatocyte, and 3) defective conjugation. Major causes of increased red blood cell destruction
include intramarrow hemolysis (eg, vitamin B12 deficiency causing ineffective erythropoiesis),
intravascular or extravascular hemolysis (eg, microangiopathic hemolytic anemia, hemolysis
from an artificial heart valve, and autoimmune hemolytic anemia [“warm,” IgG-mediated and
“cold,” IgM-mediated]), intrinsic membrane defects in red blood cells (eg, congenital spherocy-
tosis or elliptocytosis), red blood cell enzyme defects (eg, glucose-6-phosphate dehydrogenase
[G6PD] deficiency or pyruvate kinase [PK] deficiency), and hemoglobinopathies (eg, sickle cell
anemia) (Table 16–5).
TABLE 16–5 Unconjugated Hyperbilirubinemia With Hemolysis

Comment
Schistocytes present
Microangiopathic hemolytic anemia Rule out DIC, TTP, and HUS
Artificial heart valve History of valve replacement
Autoimmune hemolytic anemia Perform Coombs testing
Schistocytes absent
Intramarrow hemolysis Rule out vitamin B12 deficiency
Red blood cell membrane defects Review peripheral smear for spherocytes,
elliptocytes
Red blood cell enzyme defects Review peripheral smear for bite cells,
measure G6PD
Hemoglobinopathies Perform hemoglobin electrophoresis

DIC, disseminated intravascular coagulation; TTP, thrombotic thrombocytopenic purpura;
HUS, hemolytic uremic syndrome; G6PD, glucose-6-phosphate dehydrogenase.
TABLE 16–6 Unconjugated Hyperbilirubinemia Without Hemolysis

Newborn
Mild to moderate and transient unconjugated hyperbilirubinemia

Physiological jaundice
Breast milk jaundice
Persistent unconjugated hyperbilirubinemia
Crigler–Najjar syndrome types I (severe) and II (mild)
Child or adult
Gilbert syndrome
A variety of nonhemolytic conditions can cause an unconjugated hyperbilirubinemia

(Table 16–6). Gilbert syndrome is a benign autosomal dominant disorder in which there is a mild
defect in the uptake of bilirubin by the hepatocyte, combined with a mild defect in conjugation.
Clinical jaundice does not usually occur in the absence of concurrent disease (eg, gastroenteritis
or other mild conditions). Liver enzyme concentrations and hepatic synthetic ability are normal,
and, therefore, Gilbert syndrome is best considered a variation of normal and not a disease. On
the other hand, a congenital deficiency of UDP-glucuronyl transferase is a serious condition.
Absolute deficiency of UDP-glucuronyl transferase, which results in Crigler–Najjar syndrome
type I, will cause marked elevations in unconjugated bilirubin that will result in kernicterus in
infants if untreated. The treatment of this disease is liver transplantation. A milder deficiency of
UDP-glucuronyl transferase, Crigler–Najjar syndrome type II, may be treated with barbiturates to
stimulate production of the deficient enzyme. A transient, mild, self-limited deficiency of UDP-
glucuronyl transferase activity is common in newborns (eg, neonatal jaundice; aka icterus neona-
torum). However, if the bilirubin rises above ∼5 to 10 mg/dL in a neonate, phototherapy is used to
reduce the bilirubin concentration. One recommendation is to begin phototherapy when the bili-
rubin is 5 times the birth weight. An unconjugated, transient hyperbilirubinemia occurs in 2% to
10% of breastfed infants (ie, breast milk jaundice). In these infants, it is believed that a constituent
in the breast milk interferes with bilirubin conjugation, thereby elevating unconjugated bilirubin.
The etiologies of conjugated hyperbilirubinemia involve 2 basic mechanisms: 1) hepato-
cellular disorders with decreased transport of conjugated bilirubin into the bile canaliculus
(Table 16–3) or 2) biliary tract obstruction (Table 16–4). Moderate-to-severe acute or chronic
hepatocellular disease can produce a conjugated hyperbilirubinemia. Hepatocellular disorders
associated with impaired plasma membrane integrity and release of enzymes into the circula-
tion were discussed earlier.
Of note are 2 disorders in which there is a conjugated hyperbilirubinemia with otherwise
normal hepatic function. These are Dubin–Johnson syndrome and Rotor syndrome. Dubin–
Johnson syndrome results from dysfunction of the multidrug resistance protein 2 (MRP2) that
is a canalicular multispecific organic anion transporter (cMOAT), the gene product of ABCC2. Biliary tract obstruction
The liver is stained black in this condition. Rotor syndrome is a consequence of decreased hepatic can be intrahepatic
glutathione-S-transferase levels (hGSTA1-1). In the absence of a liver biopsy, Dubin–Johnson and or extrahepatic. The
Rotor syndromes have been distinguished by urine testing for coproporphyrins and copropor- most common cause
phyrin I that are abnormal in Dubin–Johnson syndrome. DNA testing is used progressively more of extrahepatic biliary
often to distinguish these disorders. tract obstruction after
Biliary tract obstruction can be intrahepatic or extrahepatic. Infants with a persistent the neonatal period is
conjugated hyperbilirubinemia most commonly suffer from biliary atresia or neonatal hepatitis. cholelithiasis.
The most common cause of extrahepatic biliary tract obstruction after the neonatal period is
cholelithiasis. This can be accompanied by inflammation of the gall bladder, that is, cholecystitis.
Rupture of the gall bladder will produce peritonitis that can be life-threatening. Other causes
of biliary tract obstruction include inflammation (eg, cholangitis or pancreatitis), neoplasia
(eg, pancreatic adenocarcinoma or a hepatic cancer with compression of the bile duct), postsurgical
strictures, autoimmunity (eg, primary sclerosing cholangitis [PSC]), and parasites (eg, helminths
or their ova).
Compared with the timing of elevations in the enzymes of hepatic origin following a liver
insult, elevations in bilirubin occur later. Also, while the degree of increase in the concentration
of the hepatic enzymes correlates poorly with the degree of liver injury or disease, greater eleva-
tions in the level of conjugated bilirubin do correlate with more severe degrees of liver disease.
In end-stage liver disease as found in alcoholic cirrhosis, for example, ALT and AST may only be
modestly elevated, yet the patient may exhibit intense jaundice. In such cases, portal hypertension
is frequent with ascites and esophageal varices, hemorrhoids, and splenomegaly.
SYNTHETIC FUNCTION OF THE LIVER AND DISORDERS

ASSOCIATED WITH LOW CIRCULATING CONCENTRATIONS
OF ALBUMIN, TRANSTHYRETIN, RETINOL-BINDING PROTEIN,
AND COAGULATION FACTORS
Excluding immunoglobulins, which are the products of B cells and plasma cells, the liver is the
major source of circulating proteins. On a strictly quantitative basis, albumin is a better measure
of synthetic ability than total protein. A substantial degree of hypoalbuminemia can exist, yet the
total protein may be normal or elevated because of a coexistent polyclonal or monoclonal hyper-
gammaglobulinemia. Besides liver disease, there are several other causes of hypoalbuminemia
including malnutrition and malabsorption (insufficient nutritional substrate for albumin syn-
thesis), acute inflammation where protein synthesis is redirected from albumin to acute-phase
An increase in the reactants (eg, complement proteins, C-reactive protein, mannose-binding lectin, and serum
prothrombin time (PT), amyloid A; Table 16–7), and protein loss from nephrosis or protein-losing enteropathy.
or the international In nutritionally deficient patients, nutritional replenishment can be assessed by measurement of
normalized ratio (INR) retinol-binding protein, or, more commonly, transthyretin (thyroxine-binding prealbumin). While
derived from it, can be transthyretin is commonly referred to as “prealbumin,” strictly speaking, prealbumin is a region on
a sign of serious liver a serum protein electrophoresis gel that precedes albumin. In contrast to albumin, transthyretin
dysfunction. and retinol-binding protein are not usually measured as indices of hepatic dysfunction.
TABLE 16–7 Acute-phase Reactants Synthesized in the Liver

Positive acute-phase reactants (concentrations increase with acute inflammation)
Immune-related
Complement (C′)
Mannose-binding lectin (MBL)
C-reactive protein (CRP)
Orosomucoid (alpha-1 acid glycoprotein)
Antiproteases (antienzymes)
Alpha-1 antitrypsin (A1-AT)
Alpha-2 macroglobulin (A2M)
Antioxidants
Ceruloplasmin
Coagulation factors
Fibrinogen
Factor VIII
Others
Haptoglobin
Serum amyloid A (SAA)
Plasma fibronectin
Lipopolysaccharide-binding protein (LBP)
Ferritin
Negative acute-phase reactants (concentrations decrease with acute inflammation)
Retinol-binding protein (RBP)

Transthyretin (TBPA)
Albumin
Transferrin
Assessment of clotting factor proteins through measurement of clotting factor activity tests
(such as the prothrombin time [PT]) is a useful assessment of liver synthetic function. An increase
in the PT, or the international normalized ratio (INR) derived from it, can be a sign of serious
liver dysfunction. Because the half-life of many clotting factors is much shorter than the half-life
of albumin, in cases of acute liver dysfunction, measurement of the PT can provide a more sensi-
tive index of decreased liver protein synthesis than albumin. The PT involves factors VII, X, V, II
(prothrombin), and I (fibrinogen). The half-life of factor VII is the shortest of any clotting factor
and is only 4 to 5 hours.
Despite the synthetic information provided by a prolonged PT, the severity of the coagu-
lopathy may not correlate closely with the degree of prolongation of the PT because anticoagu-
lant factor production may also be reduced with severe liver disease (eg, reduced synthesis of
antithrombin, protein S, and protein C). This means that the degree of bleeding may be less than
expected based on the degree of prolongation of the PT.
Moderate-to-serious liver disease can lead to bleeding for many reasons. Vitamin K malab-
sorption, decreased clotting factor concentrations and activity (the vitamin-K-dependent factors
are II, VII, IX, and X), and impaired clearance of fibrin-split products can all occur with liver
dysfunction. Fragments of fibrin can interfere with the formation of a stable and firm clot. With
cirrhosis, increased portal pressure can produce esophageal varices that are easily traumatized,
resulting in bleeding. Increased portal pressure can produce splenomegaly, leading to platelet
sequestration. Thrombocytopenia is not uncommon in severe liver disease. Thrombopoietin is
produced by the liver.
THE DIAGNOSIS OF VIRAL HEPATITIS

Hepatitis serologic tests are used to diagnose viral hepatitis or recognize past exposure or Hepatitis serologic tests
immunization to a virus that can cause hepatitis. The hepatitis A virus (HAV) is an RNA virus are used to diagnose viral
that commonly causes acute hepatitis and is transmitted through the fecal–oral route. Fulminant hepatitis or recognize
hepatic necrosis is possible but very rare, and chronic liver disease does not result from HAV past exposure or
infection. Total antibody to HAV can be measured, and when it is positive, there are elevations immunization to a virus
of IgM and/or IgG antibodies to HAV. Positivity for the IgM antibody to HAV indicates acute that can cause hepatitis.
or recent infection. Positivity for the HAV total antibody test does not distinguish patients with
acute infection from those with a past infection who have recovered or from immunized indi-
viduals (Table 16–8).
Acute hepatitis B virus (HBV; a DNA virus) infection is serologically first noted by the
appearance of HBV surface antigen (HBsAg). This is followed by HBV e antigen (HBeAg) and
then HBV IgM core antibody (HBc IgM antibody). During recovery, the HBsAg and HBeAg
disappear from the circulation, HBc IgM antibody converts to negative, and HBc total antibody
appears, followed by HBe antibody (HBeAb), and then HBs antibody (HBsAb).
In cases of chronic HBV infection, HBsAg remains positive. HBeAg positivity is variable, and
its presence indicates increased infectivity. HBeAb positivity is also highly variable. Immuniza-
tion results in positivity for HBsAb, but not HBcAb, as the immunogen used for immunization is
recombinant-DNA derived HBsAg.
Hepatitis C virus (HCV; an RNA virus) is the most common viral cause of chronic hepatitis
with 40% to 80% of acute infections leading to chronic hepatitis (eg, hepatic disease exceeding 6
months duration). A positive HCV antibody test does not distinguish acute from chronic infec-
tion, and it does not distinguish patients with active infection from those who have recovered.
HCV antibody positivity previously could be confirmed by the recombinant immunoblot assay
(RIBA), but RIBA testing is no longer available. However, RIBA testing has the same diagnostic
limitations as the HCV antibody testing. Evidence of active HCV infection is provided by eleva-
tions in transaminases, liver biopsy, or detection of HCV RNA by nucleic acid testing (eg, RT-
PCR, bDNA, or transcription-mediated amplification testing). Normal levels of AST and ALT do
not exclude chronic HCV infection (see Chapter 2 for illustrations of molecular methods).
Hepatitis D virus (HDV; a DNA virus) is a defective virus that requires coinfection of the host
with HBV for the expression of HDV hepatitis. HBV and HDV infection can occur concurrently
or HDV infection can be superimposed on chronic HBV infection. HDV uses the surface antigen
of HBV to form a virion. IgM antibody to HDV indicates acute infection. HDV total antibody has
TABLE 16–8 Tests for Viral Hepatitis and Selected Liver Disorders
Importance in Liver Disease
HAV total antibody Positivity indicates present or past infection with

HAV or immunization against HAV infection
HAV IgM antibody Positivity indicates acute infection with HAV
HBV surface antigen Positivity indicates acute or chronic HBV infection
HBV e antigen Positivity indicates acute or chronic HBV infection

and increased infectivity
HBV core IgM antibody Positivity indicates acute infection with HBV
HBV core total antibody Positivity indicates present or past infection with HBV
HBV e antibody Positivity indicates chronic or past infection with HBV
HBV surface antibody Positivity indicates chronic or past infection with HBV
or immunization against HBV infection
HCV antibody Positivity indicates present or past infection with HCV
HDV IgM antibody Positivity indicates acute infection with HDV
HDV antibody Positivity indicates present or past infection with HDV
Antinuclear autoantibodies Can be positive in autoimmune hepatitis
Antismooth muscle autoantibodies Can be positive in autoimmune hepatitis
Anti-LKM1 autoantibodies Can be positive in autoimmune hepatitis
Antimitochondrial autoantibodies Can be positive in primary biliary cirrhosis
Alpha-fetoprotein Marker of hepatocellular carcinoma
Ammonia Can be elevated in cases of end-stage liver disease
Serum bile acids Can be elevated in many forms of hepatocellular disease; sometimes
ordered to support the diagnosis of cholestasis of pregnancy
the same diagnostic limitations as HCV antibody; notably, active infection is not differentiated
from recovery, and acute and chronic infections are not distinguished.
SELECTED LIVER DISEASES AND LABORATORY TESTS USED

IN THE EVALUATION OF LIVER FUNCTION
Alpha-1 Antitrypsin Deficiency
In individuals with unexplained and/or early onset emphysema or liver disease, alpha-1 antitryp-
sin deficiency should be considered. Alpha-1 antitrypsin is an antiprotease that protects the lungs
from endogenous elastases, collagenases, and proteases. Deficiency of alpha-1 antitrypsin can
produce early onset panlobular emphysema. The liver disease results from the inability to release
a mutated alpha-1 antitrypsin protein.
Mutations in the Pi (protease inhibitor) gene SERPINA1 result in alpha-1 antitrypsin defi-
ciency. The normal allele is denoted as “M.” A common abnormal allele is “Z.” Homozygosity for
Z (eg, Z/Z) causes alpha-1 antitrypsin deficiency. In some forms of alpha-1 antitrypsin deficiency
where the enzyme is not synthesized at all within the hepatocyte, emphysema can develop with-
out liver disease as defective enzyme is not present in the liver.
Glycogen Storage Diseases

GSDs are disorders of glycogen production (GSD type 0) or glycogen breakdown. They are a
group of heterogenous disorders affecting the liver, skeletal muscle, and/or myocardium. GSD
types I, III, and VI can produce fasting hypoglycemia. GSD type I (von Gierke disease) results
from a deficiency of glucose-6-phosphatase (type 1a). However, variants exist as type Ib: T1 trans-
porter defects; type 1aSP: glucose-6-phosphatase stabilizing protein deficiency; type 1c: T2 beta
transporter deficiency; and type 1d: GLUT7 glucose transporter deficiency. GSD type III (Cori or
Forbes disease) is a deficiency of amylo-1,6 glucosidase. GSD type VI (Hers disease) results from
a deficiency of liver phosphorylase or phosphorylase b kinase.
Hemochromatosis
Iron overload in the absence of chronic transfusion therapy most commonly results from hemo- Iron overload in the
chromatosis type 1 (HH1) that is inherited as an autosomal recessive disorder. HH1 results from absence of chronic
mutations in the HFE gene that is encoded within the major histocompatibility complex located transfusion therapy most
on the short arm of chromosome 6. Two possible genotypes cause HH1: C282Y/C282Y or C282Y/ commonly results from
H63D. Homozygosity for H63D (H63D/H63D) does not cause clinical disease. HFE mutations hemochromatosis type 1
lead to deficient hepatic secretion of hepcidin. In turn, hepcidin deficiency permits excessive (HH1) that is inherited as
expression of ferroportin with consequent hyperabsorption of iron from the GI tract. an autosomal recessive
Increased transferrin saturation is the earliest biochemical marker of hemochromatosis. Eleva- disorder.
tions in ferritin follow. Percent transferrin saturation is the recommended screening test. Elevated
ferritin is not specific for iron overload. Ferritin is elevated as an acute-phase reactant, and ferritin
is released from the liver with disease or injury and in patients with the metabolic syndrome.
HH1 has a population frequency of ∼1 in 300, with a 5:1 to 7:1 excess of affected males
over females. The onset of symptoms is typically between 40 and 50 years of age. Iron deposition
occurs in the heart (potentially causing cardiac failure), liver (producing liver disease including
cirrhosis), endocrine organs (causing diabetes, hypopituitarism, hypothyroidism, and/or hypogo-
nadism), and skin. Arthropathy is another feature of HH1.
There are several other types of hemochromatosis in addition to HH1. HH2a results from
hemojuvelin mutations, while HH2b is caused by primary hepcidin deficiency. HH2a and HH2b
present in childhood. Mutations in the transferrin receptor 2 (TfR2) cause HH3. HH4 causes
greater iron deposition in the reticuloendothelial system than in the solid organs and liver. HH4a
is a consequence of loss of function ferroportin mutations. Aceruloplasminemia causes HH4b.
Hyperabsorption of iron from the gastrointestinal tract can occur in various forms of anemia with
ineffective erythropoiesis (eg, in cases of thalassemia and sideroblastic anemia). Hemochromatosis
can be differentiated from transfusion-related iron overload by noting whether the patient has a his-
tory of repeated transfusions. Iron overload from transfusion and hemochromatosis rarely coexist.
Wilson Disease
Wilson disease is a rare autosomal recessive disorder estimated to affect 1 in 200,000 persons.
Mutations in ATP7B result in copper overload with consequent copper deposition in the brain,
liver, kidneys, and cornea (the Kayser–Fleischer ring). ATP7B is the product of the copper-
transporting ATPase gene on chromosome 13q. ATP7B normally moves copper into the bile. In
>90% of patients with Wilson disease, the ceruloplasmin level is decreased. Copper excretion is
increased in the urine, and therefore urinary copper is a useful noninvasive test in the investiga-
tion of possible Wilson disease. Liver biopsy can provide a quantitative measure of the degree
of copper overload, as elevated hepatic copper is highly supportive of the diagnosis of Wilson
disease. The most common presentation of Wilson disease is chronic liver disease (including cir-
rhosis), but it can present as acute, fulminant hepatitis that may require liver transplantation for
survival.
Hepatocellular Carcinoma and Alpha-fetoprotein (AFP)

AFP is the major plasma protein produced by the fetal liver early in gestation. In adults, in con-
trast, AFP concentrations are normally very low. AFP may be elevated in many circumstances.
It may be transiently increased with acute liver disease or persistently increased in chronic liver
disease and cirrhosis. In patients with chronic liver disease or cirrhosis, an elevated AFP should
trigger evaluation of the patient for hepatocellular carcinoma. For this cancer, elevated AFP levels
serve as a tumor marker with a 40% to 80% sensitivity.
Acute (fulminant) hepatic Hepatic Encephalopathy and Ammonia

failure can result from a
Ammonia is an end product that results from amino acid deamination. The urea cycle captures
wide variety of insults, but
the most common causes ammonia (and thus nitrogen) for excretion by the kidney in the form of urea. Significant impair-
are acute viral hepatitis, ments in liver function produce hyperammonemia. In turn, hyperammonemia is associated with
toxins (eg, Amanita hepatic encephalopathy. A characteristic physical finding in patients with a toxic or metabolic
phalloides mushrooms), encephalopathy is asterixis (unintended jerking movements particularly of the hands when they
and poisonings (eg, are dorsiflexed).
acetaminophen).
Cholestasis of Pregnancy and Serum Bile Acid

Serum bile acid concentrations reflect the ability of the liver to remove bile acids from the circula-
tion and excrete them into the bile as part of the normal bile enterohepatic recirculation. Impaired
hepatocyte uptake or secretion of bile acids, and portosystemic shunting, can elevate serum bile
acid levels. Serum bile acids are often measured in women with cholestasis of pregnancy; other-
wise, serum bile acids are rarely measured as they add little valuable information to the standard
tests of hepatic function thus far discussed. Many argue that the diagnosis of cholestasis of preg-
nancy can be readily established without the measurement of serum bile acids.
Acute (Fulminant) Hepatic Failure

Acute (fulminant) hepatic failure can result from a wide variety of insults, but the most common
causes are acute viral hepatitis (eg, HBV and less commonly HAV), toxins (eg, Amanita phalloides
mushrooms), and poisonings (eg, acetaminophen). Other causes of acute hepatic failure include
adenovirus infection, varicella-zoster virus (VZV) infection, acute fatty liver of pregnancy, Wil-
son disease, Reye syndrome, and portal vein thrombosis.
In acute liver failure, the clinical course is rapid. Unless spontaneous recovery takes place
or liver transplantation is performed, the outcome is fatal. Acute and chronic liver failure share
many potential characteristics (Table 16–9): profound hyperbilirubinemia, coagulopathy (eg,
bleeding with a prolonged PT and thrombocytopenia), hypoproteinemia (eg, hypoalbuminemia
with edema), hypoglycemia, hyperammonemia with encephalopathy, and oliguric renal failure
(the hepatorenal syndrome resulting from, in part, splanchnic vasodilation). Chronic liver failure
TABLE 16–9 Commonly Observed Laboratory Findings in Hepatic Failure

Comment(s)
Elevated conjugated and Defects in conjugation and excretion of bilirubin

unconjugated bilirubin
Hypoalbuminemia Decreased albumin synthesis
Elevated ALT and AST Elevations of ∼100-fold with acute liver failure; rapid decline to
normal can indicate permanent loss of hepatocytes; in chronic
liver disease, ALT and AST can be normal
Hyperammonemia Impaired urea cycle
Hypoglycemia Impaired gluconeogenesis in the fasting state after glycogenolysis

has exhausted liver glycogen stores
Prolonged PT Decreased production of clotting factors, malabsorption of vitamin K,

and decreased clearance of fibrin-split products
Thrombocytopenia As a result of DIC or thrombopoietin deficiency
Anemia Bone marrow suppression leads to chronic anemia; blood loss from
esophageal varices
Elevated creatinine and BUN Decreased urine output; the hepatorenal syndrome may be present;
elevated BUN and a normal creatinine can indicate a GI bleed
DIC, disseminated intravascular coagulation; GI, gastrointestinal; PT, prothrombin time.
is also associated with intrapulmonary shunting leading to hypoxia and clubbing of the digits
(hepatopulmonary syndrome). Liver failure that occurs after 6 months of recognized liver disease
is chronic liver failure.
Cirrhosis
Cirrhosis is the outcome of any chronic disorder of the liver parenchyma or intrahepatic biliary
tract that causes continuous or repeated episodes of cellular necrosis and inflammation, followed
by subsequent episodes of repair. At some point, recurrent injury to the liver can destroy the con-
nective tissue that is the reticular structure of the liver. This results in scarring with the deposition
of increasing amounts of collagen. Bridging fibrosis can disturb intrahepatic blood flow, leading Ethanol abuse is the
to portal hypertension, with the consequent development of ascites and esophageal varices. The most common cause of
liver becomes small and firm from fibrosis, yet on physical examination the abdomen is distended cirrhosis in Westernized
because of ascites. In some patients, reopening of the umbilical vein occurs and produces peri- countries, accounting for
umbilical varices termed “caput medusa” (after the mythical Greek character Medusa). Cirrhosis 60% to 70% of cases.
can predispose to hepatocellular carcinoma. Histologically, proliferating hepatocytes appear as
regenerating nodules among the fibrotic bands. Nodules can be small (<3 mm—micronodular)
or large (>3 mm—macronodular).
Ethanol abuse is the most common cause of cirrhosis in Westernized countries, accounting
for 60% to 70% of cases. Other causes include chronic viral hepatitis (∼10%), biliary tract diseases
(∼5%-10%), and hereditary hemochromatosis (∼5%). NAFL is increasingly being recognized as a
cause of cryptogenic cirrhosis, that is, cirrhosis of otherwise unknown origin.
Patients with cirrhosis can experience severe functional liver impairment, also called “end-
stage liver disease.” Such patients can have a mixed unconjugated and conjugated hyperbilirubi-
nemia, profound hypoalbuminemia, hypoglycemia, coagulopathy (from decreased clotting factor
production, decreased clearance of fibrin-split products, and thrombocytopenia), and hyperam-
monemia.
Laboratory data can indicate the degree of liver dysfunction. However, cirrhosis remains a
clinical diagnosis until definitive diagnosis is established by the results of a liver biopsy.
Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) affects the interlobular bile ducts and is a chronic autoimmune
Primary biliary cirrhosis
biliary tract disease with obstructive jaundice. Thus, patients with PBC show a conjugated hyper-
(PBC) affects the
bilirubinemia and relative elevations in ALP exceeding the ALT and AST elevations. ALT and interlobular bile ducts and
AST can be normal. is a chronic autoimmune
Approximately 95% of patients with PBC are positive for anti-mitochondrial antibodies biliary tract disease with
(AMA). Portal inflammation and progressive scarring can progress to liver failure requiring liver obstructive jaundice.
transplantation. The condition most often affects women between the ages of 40 and 50 years.
Primary Sclerosing Cholangitis

PSC affects the larger bile ducts. Men are more commonly affected than women (70:30 ratio),
with a mean age at onset near 40 years. Tests for AMA are negative in patients with PSC. Inflam-
matory bowel disease, such as Crohn disease or ulcerative colitis, is identified in about 75% of
patients with PSC. The definitive diagnosis of PSC is made by liver biopsy.
APPROACH TO THE PATIENT WITH LIVER DISEASE

One reasonable approach to the evaluation of liver function is to first consider the bilirubin con-
centration (Figure 16–1 and Tables 16–3 to 16–6). If there is an unconjugated hyperbilirubine-
mia, possible hemolysis should be investigated. If this is absent, other causes of an unconjugated
hyperbilirubinemia need to be considered, such as neonatal jaundice, Gilbert syndrome, and
Crigler–Najjar syndrome.
If there is a conjugated hyperbilirubinemia, the liver enzymes can be used to separate
hepatocellular disease (eg, predominant elevations in ALT and AST) from biliary tract disease
Hyperbilirubinemia present
Unconjugated Conjugated Normal bilirubin
Evidence of hemolysis ALT & AST increase > ALP increase Normal enzymes
Yes∗ No Yes No
Identify specific Identify specific Identify specific Identify Dubin-Johnson

hemolytic non-hemolytic hepatocellular specific biliary or Rotor
etiology etiology etiology tract etiology syndrome
FIGURE 16–1 One approach to the evaluation of liver function. (*) Increased LD, decreased
haptoglobin; with or without an abnormal peripheral smear.
(eg, predominant elevations in ALP, or if measured, GGT and 5′-NT). Disorders are then
investigated based on their relative frequency and whether the disease is acute or chronic. Not
noted in Figure 16–1 is end-stage liver disease in which there can be significant elevations in both
the conjugated and unconjugated fractions. In the absence of hyperbilirubinemia, the focus on
liver dysfunction becomes the pattern of enzyme elevation.
REFERENCES
Dufour DR, et al. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory
tests. Clin Chem. 2000;46:2027–2049.
Dufour DR, et al. Diagnosis and monitoring of hepatic injury. II. Recommendations for use of laboratory
tests in screening, diagnosis, and monitoring. Clin Chem. 2000;46:2050–2068.
Dufour DR, et al. The National Academy of Clinical Biochemistry, laboratory medicine practice guidelines,
laboratory guidelines for screening, diagnosis and monitoring of hepatic injury. <http://www.aacc.org/
members/nacb/Archive/LMPG/HepaticInjury/Pages/HepaticInjuryPDF.aspx>. Accessed May 28, 2009.
Fix OK, Kowdley KV. Hereditary hemochromatosis. Minerva Med. 2008;99(December):605–617.
Hogarth DK, Rachelefsky G. Screening and familial testing of patients for alpha 1-antitrypsin deficiency.
Chest. 2008;133:981–988.
Langner C, Denk H. Wilson disease. Virchows Arch. 2004;445:111–118.
Limdi JK, Hyde GM. Evaluation of abnormal liver function tests. Postgrad Med J. 2003;79(June):307–312.
Mallory MA, et al. Abnormal liver test results on routine screening. How to evaluate, when to refer for a
biopsy. Postgrad Med. 2004;115:53–56, 59–62, 66.
C H A P T E R
Pancreatic Disorders
David N. Alter and Michael Laposata 17
LEARNING OBJECTIVES
1. Understand the differences between acute and chronic pancreatitis and
the laboratory test results used to establish the diagnosis of each.
2. Learn the clinical, laboratory, and radiographic abnormalities in patients
with cancer of the pancreas.
3. Learn the clinical and laboratory criteria for the diagnosis of diabetes
mellitus, gestational diabetes mellitus, and hypoglycemia.
4. Identify the different islet cell tumors and learn their associated laboratory
test results.
CHAPTER OUTLINE
Introduction 371 Diabetes 378
Acute Pancreatitis 372 Fasting Blood Glucose 378
Chronic Pancreatitis 374 Oral Glucose Tolerance Test 378
Pancreatic Tumors 375 Random Blood Glucose 378
Exocrine Pancreas Neoplasms 375 Disease Monitoring 379
Endocrine Pancreas Neoplasms 375 Hemoglobin A1c 379
Diabetes Mellitus 376 Other Markers 379
Prediabetes 377 Gestational Diabetes Mellitus 380
Hypoglycemia 381
INTRODUCTION
Disorders involving the pancreas are generally divided into 2 categories. One group includes
diseases of the exocrine portion of the pancreas, which secretes digestive enzymes into the gas-
trointestinal tract. The other category includes the disorders of the endocrine portion of the
pancreas, which contains beta cells for secretion of insulin, alpha cells for secretion of glucagon,
and delta cells for secretion of somatostatin. The cells that secrete hormones are arranged in islets
within the exocrine pancreas.
The most frequently encountered disorders of the exocrine pancreas are pancreatitis and
pancreatic neoplasms (usually cancer). Pancreatitis may be acute, or chronic with recurrent bouts
of acute pancreatitis. Pancreatic tumors of the exocrine pancreas almost always originate in the
pancreatic ductal epithelium. The major disease of the endocrine pancreas is diabetes mellitus
(DM). Several neoplasms are also associated with the endocrine pancreas but are much rarer than
those associated with the exocrine pancreas.
371
372 CHAPTER 17 Pancreatic Disorders
ACUTE PANCREATITIS
Description
Acute pancreatitis is Acute pancreatitis is a potentially lethal disorder associated with intracellular activation of diges-
a potentially lethal tive enzymes in the pancreas. This results in autodigestion of the pancreatic tissue by the powerful
disorder associated with enzymes normally secreted into the gastrointestinal tract to degrade ingested foods. The damage
intracellular activation to the pancreas can produce inflammation, edema, necrosis, hemorrhage, and liquefaction, and
of digestive enzymes in may obstruct the pancreatic duct and block the flow of pancreatic enzymes into the gastrointes-
the pancreas. This results tinal tract. The obstruction further enhances the progression of acute pancreatitis. Clinically, a
in autodigestion of the bout of acute pancreatitis is characterized by midepigastric pain frequently radiating to the back,
pancreatic tissue by nausea, and vomiting.
the powerful enzymes The cause of acute pancreatitis in the majority of the cases is either alcohol abuse or gall-
normally secreted into the
stones. There are, however, other causes, such as hypertriglyceridemia, hypercalcemia, selected
gastrointestinal tract to
infections, obstructing pancreatic tumors, and trauma to the pancreas. Hereditary forms of acute
degrade ingested foods.
pancreatitis have also been described due to mutations in the trypsinogen gene or the trypsin
inhibitor gene. In addition, many medications have been associated with the development of
acute pancreatitis. Selected examples are asparaginase, azathioprine, estrogens, furosemide, sul-
fonamides, tetracycline, and thiazide diuretics. The mechanism of pancreatic injury following
ingestion of these medications may be related to hypersensitivity to the drug or accumulation of a
toxic drug metabolite in the pancreas. In about 20% of cases of acute pancreatitis, a specific cause
cannot be identified. This is known as idiopathic acute pancreatitis.
Diagnosis
A large number of potential markers (carboxyester lipase, carboxypeptidase, trypsin, trypsino-
gen-2, pancreatitis-associated protein, trypsinogen activation peptide, C-reactive protein, and
tumor necrosis factor) are discussed in the literature as possible for the diagnosis of acute pan-
creatitis; however, none of them are typically available in most hospital laboratories. In fact, acute
pancreatitis is not solely diagnosed by a biochemical marker but rather by a combination of his-
tory, clinical presentation, and radiologic and laboratory findings (Table 17–1).
Nonspecific laboratory findings include, but are not limited to, aminotransferase elevations,
mild-to-moderate leukocytosis with a shift toward immature forms, hyperglycemia, mild hyper-
bilirubinemia, and a decreased serum calcium level.
More specific biochemical tests used for the diagnosis are amylase and lipase. Historically,
an amylase level greater than 3× the upper limit of normal was considered diagnostic for acute
pancreatitis, and lipase findings were helpful but not as useful. The problem with lipase was the
existence of a number of different assay methods and no standardization between them. This has
changed. Current assays for lipase are more standardized, and the lipase assay has several charac-
teristics that make it a better marker for acute pancreatitis.
Temporally, in acute pancreatitis, amylase will rise and fall over a shorter period of time
(rise over 6-24 hours, peak at 48 hours, and normalize in 5-7 days). Lipase has different tem-
poral kinetics (rise over 4-8 hours, peak at 24 hours, and normalize in 8-14 days). Therefore, it
TABLE 17–1 Laboratory Evaluation for Acute and Chronic Pancreatitis and
Pancreatic Carcinoma
Disorder Test Expected Result
Acute pancreatitis Serum amylase Increased

Serum lipase Increased
Amylase/creatinine clearance Increased
Chronic pancreatitis Serum amylase Increased, normal, or decreased

Serum lipase Increased, normal, or decreased
Amylase/creatinine clearance Increased, normal, or decreaseda
a
In chronic pancreatitis, the amylase/creatinine clearance ratio can be increased even when the serum amylase is normal
or only slightly elevated. The amylase/creatinine clearance ratio is: (urine amylase [U/L] × serum creatinine [mg/L])/(serum
amylase [U/L] × urine creatinine [mg/L]) × 100.
CHAPTER 17 Pancreatic Disorders 373
is more possible to miss an amylase elevation than a lipase elevation. In addition, despite the
fact that neither marker is organ specific for the pancreas, lipase has relatively greater pancreas
specificity than amylase. There are a larger number of conditions that can result in non-pancre-
atitis hyperamylasemia than there are that can result in hyperlipasemia. Conditions associated
with non-pancreatitis increases in both amylase and lipase include biliary disease, intestinal
obstruction, pancreatic pseudocyst, and renal impairment. Clinical conditions associated with
elevations in amylase without a corresponding increase in lipase include macroamylasemia,
ruptured ectopic pregnancy, salivary gland disease, and abdominal and thoracic malignancies.
Renal failure is the most common extrapancreatic condition associated with an elevated serum
lipase level. About 80% of patients with renal failure have lipase levels 2 to 3 times the upper
limit of the reference range, and about 5% have an elevation more than 5 times the upper limit
of normal.
Amylase elevation greater than 3× the upper limit of normal is the cutoff often cited for the
diagnosis of acute pancreatitis. Test characteristics for amylase in the diagnosis of acute pan-
creatitis range from 45% to 85% for sensitivity with specificities ranging from 90% to 99%. Test
characteristics for lipase elevations have sensitivities and specificities greater than 95% for the
diagnosis of acute pancreatitis.
In cases of hyperamylasemia where the clinical presentation does not support the diagnosis
of acute pancreatitis, a urine amylase level may have clinical utility.
A urine amylase determination may be helpful in diagnosing pancreatic disorders, especially
when the serum amylase level is normal or slightly elevated. As a general rule, urine amylase rises
within 24 hours after an increase in serum amylase, and remains high for 7 to 10 days after the
serum level returns to normal. Renal excretion of amylase depends on the glomerular filtration
rate and, consequently, the urine amylase correlates with the creatinine clearance (CC). In acute
pancreatitis, the clearance of amylase into the urine may be increased compared with creatinine,
resulting in an increased (amylase/creatinine clearance [A/CC]) ratio. The A/CC ratio is deter-
mined using the following formula: In acute pancreatitis,
the clearance of amylase
Urine amylase (U/L) × Serum creatinine (mg/L) into the urine may be
× 100 increased compared with
Serum amylase (U/L) × Urine creatinine (mg/L)
creatinine, resulting in
an increased (amylase/
Determination of the A/CC ratio involves simultaneous collection of serum and urine specimens
creatinine clearance
but does not require a timed or complete 24-hour urine collection. The A/CC ratio becomes
[A/CC]) ratio.
abnormal 1 to 2 days after an elevation of the serum amylase, and typically remains abnormal for
as long as the urine amylase is high. Like the urine amylase level, the A/CC ratio remains elevated
for 7 to 10 days after the serum amylase level returns to normal.
Macroamylasemia is an established cause for an elevated serum amylase value. Macroamylase
is a complex of alpha-amylase and other molecules, which may be proteins or carbohydrates.
Because of its large molecular size, macroamylase is not filtered by the glomerulus in the kid-
ney. Consequently, it accumulates in the serum, and produces a chronically elevated serum
amylase level. The presence of macroamylase has been shown to account for an elevated serum
amylase level in 1% to 3% of patients. Because macroamylase does not enter the urine, the
urine amylase level is normal or low, unlike the situation in acute and chronic pancreatitis in
which the urine amylase level is usually elevated along with the serum activity. Thus, patients
with macroamylasemia have a combination of elevated serum amylase levels and normal or low
urine amylase levels.
Both pancreatic and salivary amylase have more than 1 isoenzyme alpha-amylase can be
pancreatic or salivary. Amylase can be separated into its component isoenzymes by selective
enzymatic or chemical inhibition and by electrophoresis. In acute pancreatitis, there is an increase
in pancreas-derived isoenzymes in almost all patients, but isoenzyme analysis is rarely required
for diagnosis.
A number of laboratory tests and computed tomography may be useful to assess prognosis in
patients with acute pancreatitis. One system to assign prognosis in acute pancreatitis is the sim-
plified Glasgow criteria. Features associated with a worse prognosis include age >55 years, white
blood cell count >15,000/μL, LD >600 U/L, glucose >180 mg/dL, albumin <3.2 g/dL, calcium
<8 mg/dL, arterial Po2 <60 mm Hg, and BUN >45 mg/dL.
CHRONIC PANCREATITIS
Description
Following an attack of Following an attack of acute pancreatitis, the patient may experience a complete recovery, have
acute pancreatitis, the an additional recurrence without permanent damage to the pancreas, or suffer multiple recur-
patient may experience a rences leading to chronic pancreatitis and significant damage to the organ. In chronic pancreati-
complete recovery, have tis, the cells that generate the digestive enzymes are destroyed and replaced with scar tissue, and
an additional recurrence the pancreatic ducts become dilated and filled with precipitated protein. Chronic pancreatitis
without permanent can be divided into chronic calcifying pancreatitis and obstructive pancreatitis. Since chronic
damage to the pancreas, disease follows from recurrence of acute pancreatitis, chronic pancreatitis has various causes in
or suffer multiple adults. In the United States, the majority of cases of chronic pancreatitis are due to prolonged
recurrences leading to excessive alcohol consumption. Malnutrition-induced pancreatitis is more common in underde-
chronic pancreatitis and
veloped areas of the world. In children, the most common cause of chronic pancreatitis is cystic
significant damage to the
fibrosis (see Chapter 7).
organ.
Diagnosis
The diagnosis of chronic pancreatitis may be challenging because the disease can evolve sub-
clinically over an extended period. The patient with chronic pancreatitis often has impaired glu-
cose tolerance (IGT) or, in severe cases, DM. Additional manifestations include abdominal pain,
weight loss, pancreatic calcifications on x-ray, and steatorrhea. The sensitivity of laboratory tests
to diagnose chronic pancreatitis depends on the extent of pancreatic tissue destruction and the
length of time over which the damage has occurred.
An elevated serum amylase level is much less informative to make a diagnosis of chronic
pancreatitis than it is in the diagnosis of acute pancreatitis. In about one half of the patients with
chronic pancreatitis, the serum amylase level remains within the normal range. In other patients
with the disorder, the values may be borderline or only slightly elevated, raising the possibil-
ity of a nonpancreatic cause for the elevated amylase. In chronic pancreatitis, the urine amy-
lase level may be elevated when the serum amylase is within the normal range or only slightly
elevated. Measurement of a 72-hour fecal fat provides an index of pancreatic exocrine function.
It is increased in severe chronic pancreatitis. However, the fecal fat test is neither sensitive nor
specific. More recently, measurement of fecal elastase (decreased in chronic pancreatitis) and
serum levels of trypsinogen (decreased in chronic pancreatitis) have been used as additional
tests of pancreatic function.
The bentiromide test is a noninvasive test for assessing pancreatic function in patients sus-
pected to have chronic pancreatitis. The test is based on the hydrolysis by chymotrypsin of a
synthetic tripeptide, N-benzoyl-l-tyrosyl-p-aminobenzoic acid. The tripeptide, variously called
bentiromide, NBT-PBA, or BTP, is administered orally, along with a test meal to stimulate pan-
creatic secretion. Chymotrypsin cleaves the p-aminobenzoic acid (PABA) molecule from the
bentiromide in the duodenum. The PABA moiety is absorbed into the portal circulation, conju-
gated in the liver, and excreted by the kidneys as an arylamine. In the bentiromide test, the aryl-
amines are quantitated in a 6-hour urine specimen, with the time started after the oral intake of
bentiromide and the test meal. Decreased excretion (<50% of the test dose) suggests decreased
absorption from the duodenum, which can occur with deficient activity of pancreatic chymo-
trypsin. The sensitivity of the test for diagnosis of chronic pancreatitis depends on the severity
of the disease, with greater sensitivity of the test correlating with greater disease severity. Many
nonpancreatic conditions, especially diseases of the kidney, are associated with a false-positive
test result by decreasing the conjugation and/or excretion of the PABA metabolite in the urine.
Conversely, a number of drugs (acetaminophen, lidocaine, procainamide, sunscreens containing
PABA, and pancreatic enzyme supplements, as examples) may produce a falsely normal result in
a patient with chronic pancreatitis, because these products can increase the amount of arylamine
in the urine.
Imaging studies including abdominal plain films may demonstrate calcifications. Ultra-
sound and computed tomography scans are relatively sensitive and specific. Duodenal intuba-
tion using endoscopic retrograde cholangiopancreatography (ERCP), with injection of x-ray
contrast medium into the common bile duct and pancreatic ducts, is the most sensitive test,
but the test itself may induce pancreatitis and should therefore be reserved for selected cases.
More recently endoscopic ultrasound has gained favor, and it is equally sensitive and specific for
chronic pancreatitis as ERCP.
PANCREATIC TUMORS
Exocrine Pancreas Neoplasms
Description
Masses within the pancreas can be either nonneoplastic or neoplastic. The nonneoplastic masses Pancreatic cancer affects
are almost always cystic. However, both benign and malignant pancreatic tumors may be cystic. more than 30,000 adults
A cyst can be congenital from abnormal development, but more often, it is a collection of pancre- in the United States
atic secretions and tissue debris following bouts of pancreatitis, and is known as a pseudocyst. In annually and is usually
contrast to true cysts, pseudocysts lack an epithelial lining. Pancreatic cancer affects more than rapidly fatal.
30,000 adults in the United States annually and is usually rapidly fatal. The great majority of these
tumors arise in the exocrine pancreas and histologically are ductal adenocarcinomas.
Diagnosis
CA 19-9 is the most widely used pancreatic tumor marker. CA 19-9 antigen is present in the
normal adult and fetal pancreas, and it is also found in the esophagus, stomach, small intestine,
gallbladder, bile duct, and salivary glands. For the diagnosis of pancreatic cancer, the reported
sensitivity ranges from 70% to 92% with a specificity that ranges from 68% to 92%. The marker’s
sensitivity is proportional to tumor size. Measuring the level of the CA 19-9 may be useful in
patients with pancreatic cancer. In patients with early stage tumors, the CA 19-9 level is often
normal. Therefore, the marker is of little value as a screening test. In patients with more advanced
tumors, the CA 19-9 level is often elevated, and this finding may be helpful in suggesting a diag-
nosis of pancreatic cancer. CA 19-9 is most useful as an aid to monitor the patient response to
therapy. However, CA 19-9 is not specific to pancreatic cancer and may be elevated in other types
of gastrointestinal cancers and in some nonneoplastic disorders as well. Of note, CA 19-9 requires
the presence of the Lewis blood group antigen for it to be expressed. Therefore, CA 19-9 will be
naturally undetectable in 7% to 10% of the population.
Endocrine Pancreas Neoplasms

Islet Cell Tumors
Islet cell tumors, which may occur as a single mass or multiple masses, may be associated with
hyperfunction of specific hormone-secreting cells within the islets of Langerhans of the pan-
creas. Some islet cell tumors are nonfunctional, and therefore may present only with symptoms
of a mass lesion. Radiographic studies to identify the tumor and permit surgical removal are an
important part of the evaluation of the patient for an islet cell tumor.
• Beta cell tumors are also known as insulinomas and are clinically significant when they
produce enough insulin to induce hypoglycemia. The laboratory studies used in the
detection of insulinoma include plasma glucose, C-peptide, insulin, and the insulin to
glucose ratio. Hypoglycemia is discussed in detail below.
• Tumors of the pancreatic islets that secrete gastrin are known as gastrinomas. However,
the most common site of a gastrinoma is the duodenum. An elevated serum gastrin level
from a gastrin-secreting tumor is associated with the development of peptic ulcer disease
because gastrin stimulates acid secretion as well as watery diarrhea and malabsorption. This
constellation of clinical and laboratory findings constitutes Zollinger–Ellison syndrome.
Patients with peptic ulcer disease who do not have a Helicobacter pylori infection or a
history of nonsteroidal anti-inflammatory drug use may have Zollinger–Ellison syndrome
and should be evaluated for a gastrinoma. Patients with Zollinger–Ellison syndrome may
also have multiple endocrine neoplasia I, with islet cell tumors that produce a variety of
hormones. The secreted hormones found, in descending order of frequency, are: gastrin,
insulin, serotonin, and vasoactive intestinal peptide (VIP), which is associated with watery
diarrhea. The diagnosis of a gastrinoma is based on the finding of an elevation in serum

gastrin while fasting in association with increased gastric acid secretion.
• Tumors of the alpha cells of the pancreatic islets, also known as glucagonomas, are
associated with elevated serum levels of glucagon. These tumors can be associated with a
characteristic migratory erythema, as well as glucose intolerance, weight loss, deep vein
thrombosis, and depression.
• Tumors of the delta cells of the endocrine pancreas are known as somatostatinomas.
These tumors are typically associated with diabetes-related symptoms, diarrhea, steatorrhea,
cholelithiasis, and weight loss. These tumors are most often located in the duodenum or
jejunum, rather than in the pancreas.
• Some islet cell tumors produce vasoactive intestinal peptide (VIP). These tumors, known
as a VIPomas, induce a syndrome of watery diarrhea, hypokalemia, hypochlorhydria,
and acidosis (WDHHA syndrome). Serum VIP levels are elevated in patients with VIPomas.
DIABETES MELLITUS
Description
DM represents a heterogenous group of disorders with the common feature of hyperglycemia
due to defects in insulin secretion, insulin action, or a combination of these 2 factors. Table 17–2
shows the criteria for the diagnosis of DM. Disease sequelae are not directly related to the degree
of hyperglycemia as much as they are related to the acute and chronic effects of hyperglycemia on
end-organ processes.
For the acute inpatient population hyperglycemia is associated with increased morbidities
and poorer prognoses for almost every acute disease process, most notably, but far from limited
to, sepsis, acute coronary syndromes, and surgical recovery. As a result, all hospitals have now
implemented policies and procedures to minimize inpatient hyperglycemia for both the diabetic
and nondiabetic patients under the umbrella term, “tight glycemic control.”
Chronic hyperglycemia has been strongly associated with the development of polyneuropa-
thy, nephropathy, and retinopathy, as well as a reduced ability to fight infections. Almost 13%
of Americans have DM, of which 40% are unaware of their disorder. A significant fraction will
Almost 13% of Americans
already have some degree of nephropathy, neuropathy, and/or retinopathy when they are first
have DM, of which 40%
diagnosed with DM. Importantly, many of the complications of diabetes can be avoided through
are unaware of their
early diagnosis and aggressive management.
disorder. A significant
fraction will already The American Diabetes Association (ADA) sponsored the formation of an Expert Commit-
have some degree of tee on the Classification and Diagnosis of Diabetes Mellitus to establish guidelines. The World
nephropathy, neuropathy, Health Organization (WHO) adopted these guidelines, and further refined the diagnostic criteria
and/or retinopathy when for gestational diabetes.
they are first diagnosed Central to the ADA guidelines are etiology-based rather than treatment-based definitions
with DM. for type 1 and type 2 DM and other disorders of glucose regulation (Table 17–3). The treatment
TABLE 17–2 Criteria for Diagnosis of Diabetes Mellitus

Hemoglobin A1c (HbA1c) greater than or equal to 6.5%. The method used should be certified and standardized
to the diabetes control and complications trial (DCCT) assay
Or
Fasting plasma glucose greater than or equal to 126 mg/dL (7.0 mmol/L). Fasting is defined as no caloric intake
for at least 8 h
Or
A 2-h plasma glucose value greater than 200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test.
The tests should be performed using a glucose load containing the equivalent of 75 g of anhydrous
glucose dissolved in water
Or
A patient with classic symptoms of hyperglycemia or hyperglycemic crisis and a random plasma glucose
value greater than 200 mg/dL (11.1 mmol/L)
TABLE 17–3 American Diabetes Association Classification of Diabetes Mellitus

Classification Pathogenesis
Type 1 diabetes Absolute deficiency of insulin secretion, usually due to immune-

mediated beta-cell destruction
Type 2 diabetes Varying degrees of insulin resistance; even if there is increased

plasma insulin, it is insufficient to compensate for the resistance
Other specific types of diabetes Heterogenous causes, subclassified as: genetic defects of
beta-cell function, genetic defects in insulin receptors, exocrine
pancreatic disease, drugs or chemicals toxic to islet cells or that
antagonize insulin, infectious destruction of islet cells, uncommon
forms of immune-mediated diabetes, or other endocrine diseases
that impair glucose regulation
Gestational diabetes Various causes, including unrecognized type 1 diabetes

and subclinical incipient type 2 diabetes
can vary with the disease course. For instance, patients termed “noninsulin-dependent” diabetics
may eventually require insulin to control their hyperglycemia. The categories type 1 and type 2
diabetes are now designated by Arabic, rather than by Roman numerals. The majority of patients
(90%-95%) have type 2 diabetes.
Diagnosis
Hyperglycemia, by itself, does not establish a diagnosis of diabetes. A diagnosis of diabetes depends Hyperglycemia, by itself,
on whether normal physiologic homeostasis and normoglycemia recur after a glucose elevation, does not establish a
and the normal glucose level is maintained. Criteria have been established by the ADA and WHO diagnosis of diabetes.
to diagnose DM determined by age (pediatric vs adult) and pregnancy status (pregnant or not). A diagnosis of diabetes
Specifics between the ADA and WHO may differ, but the theme remains the same. In the United depends on whether
States, ADA criteria are used, and external to the United States, WHO criteria may be used. Table normal physiologic
17–4 shows high-risk individuals for whom diabetes screening is recommended by the ADA. homeostasis and
normoglycemia recur
after a glucose elevation,
Prediabetes and the normal glucose
level is maintained.
Although not meeting criteria for diabetes, an intermediate group of subjects exist whose glu-
cose levels are too high to be considered normal but not high enough to be classified as diabetic
(Table 17–2). Patients with impaired fasting glucose (IFG) and/or impaired glucose tolerance
(IGT) have a prediabetic condition, indicating a high risk for development of DM. In the absence
of pregnancy, IFG and IGT are not clinical entities in their own right, but instead identify patients
at risk for DM and its cardiovascular complications. Loss of 5% to 10% of body weight, exercise,
and treatment with appropriate medications are measures taken to prevent or delay the onset of
DM in patients with “prediabetes.” Of note, the “impaired criteria” have changed as more data
have become available, and it is possible that they will change again, thereby shifting formerly
“prediabetic” individuals into the “diabetic” category.
TABLE 17–4 High-risk Individuals for Whom Diabetes Mellitus Screening

Is Recommended by the American Diabetes Association
Individuals of the following ancestry: African, Asian, Hispanic, Native American, and Pacific Islander
Mothers with newborns >9 lb or history of gestational diabetes mellitus
Individuals with hypertension ≥140/90, HDL cholesterol ≤35 mg/dL, or triglycerides ≥250 mg/dL
Individuals with a history of impaired fasting glucose or impaired glucose tolerance; HbA1c ≥5.7%
Obese individuals weighing ≥120% of ideal body weight
Individuals with first-degree relatives with diabetes mellitus

HDL, high-density lipoprotein.
As mentioned before, about half of all Americans with type 2 DM go undiagnosed and suf-
fer from preventable complications of the disease. Therefore, the ADA has advocated screening
everyone over 45 years old with a fasting plasma glucose (FPG) test, with a repeat test every
3 years if the results are negative. Screening of groups with high risk for DM has been proposed
for individuals <45 years old (Table 17–4).
DIABETES
Fasting Blood Glucose
The most commonly used The most commonly used laboratory test for the diagnosis of diabetes is the FPG, measured after
laboratory test for the at least 8 to 12 hours of fasting. The patient can drink water while fasting, but should abstain
diagnosis of diabetes is from eating, smoking, or taking medications. Acute illness, surgery, and hospitalization within
the FPG, measured after the previous 8 weeks are relative contraindications to testing, as false-positive results can arise in
at least 8 to 12 hours of these situations. The threshold of 125 mg/dL for the FPG test is lower than that used previously
fasting. (140 mg/dL). The 125 mg/dL threshold corresponds to an epidemiological breakpoint, above
which the risk for retinopathy and nephropathy increases dramatically. Patients with IFG have a
FPG ranging from 110 to 125 mg/dL. This group is considered at risk for subsequent development
of DM, as well as for cardiovascular disease.
Blood glucose levels in some fashion have been used for a long time as the diagnostic cri-
teria for diabetes. However, in 2009, another biochemical marker was added: hemoglobin A1c
(HbA1c). HbA1c had been in use for about 2 decades as a prognostic marker of chronic hyper-
glycosylation because it was shown that the degree of its elevation is directly proportional to
the degree of end-organ hyperglycosylation, and, therefore, directly related to the degree of end-
organ damage. In 2009, the International Expert Committee on the role of HbA1c in the diagno-
sis of diabetes concluded that the HbA1c assay may be a better means of diagnosing diabetes than
the measurement of blood glucose. Up to that point, HbA1c assay methods and results showed
wide variation. However, as the assays improved and variation in results from method to method
decreased, it gained utility as a diagnostic marker of diabetes. The committee recommends that
a diagnosis of diabetes can be made if the HbA1c level is ≥6.5% (see below and Table 17–2).
However, they also recommend that the diagnosis of diabetes should be confirmed with a repeat
HbA1c test if the first one is greater than 6.5%, unless the patient already has clinical symptoms of
diabetes or a blood glucose level greater than 200 mg/dL, in which case a single value greater than
6.5% is adequate to establish the diagnosis.
Oral Glucose Tolerance Test

If the clinical picture If the clinical picture merits further testing for diabetes in a patient with a FPG ≤125 mg/dL, an
merits further testing for oral glucose tolerance test (OGTT) is indicated. The patient should have a regular diet during
diabetes in a patient with the preceding 3 days, with a carbohydrate intake of at least 100 g per day. The patient’s activity
a FPG ≤125 mg/dL, an oral should be unrestricted, and only severe illness or hospitalization represents relative contrain-
glucose tolerance test dications. Minor illnesses with gastrointestinal manifestations are not significant. The glucose
(OGTT) is indicated. bolus for nonpregnant adults is 75 g of anhydrous glucose dissolved in 10 to 12 oz of water or
a preformulated flavored drink containing 75 g of glucose, such as glucola. For children, 1.75 g
of glucose per kilogram weight is administered, up to a maximum of 75 g. The bolus should be
consumed over 5 minutes. The testing protocol has been simplified from previous versions to
include only 2 specimens: a fasting specimen and one 2 hours after the bolus. A 2-hour postbolus
plasma glucose level of ≥200 mg/dL is diagnostic of DM. A 2-hour postbolus plasma glucose
level of ≥140 mg/dL but <200 mg/dL is found in patients with IGT. Patients in this group, like
those with IFG, are considered at risk for subsequent development of DM as well as cardiovas-
cular disease.
Random Blood Glucose

A random plasma glucose level of ≥200 mg/dL, combined with symptoms of DM (polyuria,
polydipsia, and unexplained weight loss), also can be used to establish a diagnosis of DM.
These criteria do not depend on the time since the last meal, but the test should not be done
when the patient is acutely ill.
DISEASE MONITORING
Hemoglobin A1c
The measurement of glycohemoglobin, specifically HbA1c, is essential for monitoring the The measurement of
success of therapy for patients with DM. HbA1c is formed by the nonenzymatic linkage of glycohemoglobin,
glucose to hemoglobin. Glucose enters the red blood cell, and it becomes bound to hemo- specifically HbA1c, is
globin. An aldimine is first formed that then undergoes an Amadori rearrangement to form essential for monitoring
a stable ketoamine, which persists for the life span of the red blood cell (typically 120 days). the success of therapy
The HbA1c concentration does not exhibit the wide diurnal fluctuations that occur with blood for patients with DM.
glucose. The blood glucose concentration varies substantially with exercise, food ingestion, HbA1c is formed by the
and many other factors. The rate of formation of HbA1c is directly proportional to the glu- nonenzymatic linkage of
glucose to hemoglobin.
cose concentration in the blood. Because of this, the HbA1c concentration is a reflection of
the glucose values over the preceding 8 to 12 weeks. HbA1c is primarily used for monitoring
long-term glycemic status and to determine whether a diabetic patient has achieved adequate
metabolic control.
In diabetic patients, the retinopathy incidence increases substantially at HbA1c values
between 6.0% and 7.0%. There is a low prevalence of retinopathy at HbA1c levels less than 6.5%.
A HbA1c value of <7% is widely recommended. The ADA recommends measurement of HbA1c
at least twice per year in all persons with DM. As noted above, a report from the International
Expert Committee in 2009 on the role of HbA1c concludes that large volumes of data from diverse
populations provide strong justification for assigning a reproducible HbA1c level of >6.5% as
adequate for the diagnosis of diabetes.
Blood specimens for all these tests should be collected in gray-top tubes, as the sodium fluo-
ride anticoagulant will inhibit glycolysis. Without sodium fluoride, the metabolism of glucose
by the white blood cells in a specimen can lower the plasma glucose levels by 5% to 7% per
hour. Serum levels are comparable to plasma levels if the serum is separated from the cells within
1 hour and testing is performed within 8 hours. Capillary blood glucose levels are approximately
10 mg/dL lower than plasma levels when fasting, but are equal to or higher than plasma levels
after a glucose load. For the diagnosis of DM from a circulating glucose concentration, a plasma
sample is the preferred specimen.
Point-of-care glucose testing is widely available in inpatient, outpatient, and home use popu-
lations. It is a very convenient and a simple to use modality for monitoring blood glucose levels
before meals, assessing potential bouts of hypoglycemia or hyperglycemia, and for monitoring
compliance with personalized diabetic regimens. These devices are not recommended to establish
diagnosis of diabetes.
Other Markers
Considerable attention is now focused on the detection of autoantibodies as a screening tool
for asymptomatic individuals with a strong family history of type 1 DM. The presence of auto-
antibodies to 2 or more of the following—glutamic acid decarboxylase (GAD65), islet tyrosine
phosphatase (ICA512), or insulin—is a strong predictor of progression to type 1 DM (greater
than 50%). It remains to be shown, however, whether early intervention can slow or prevent the
subsequent onset of disease. Therefore, the ADA does not currently advocate screening for dia-
betes with these tests.
An important aspect of the ADA classification system is the prognosis for the patients based
upon the etiology of DM in a given patient. DM from certain causes, such as drug use or endo-
crine tumors, may be completely reversible. DM from other causes such as insulin receptor defects
are not reversible, and are often difficult to manage. DM categorized as “other specific types of
diabetes” in Table 17–3 are much rarer than type 1 or type 2 DM. These types deserve some con-
sideration whenever a new diagnosis of DM is made, as shown in Table 17–5.
TABLE 17–5 Laboratory Evaluation for Selected Other Causes of Diabetes Mellitus
Etiology Test(s) for Evaluation
Exocrine pancreatic disease Amylase, lipase
Cushing syndrome 24-Hour urine-free cortisol
Glucagonoma Plasma glucagon
Hyperthyroidism Thyroid-stimulating hormone (TSH)

Hemochromatosis Iron, ferritin, total iron-binding capacity
The treatment goals for patients with DM include the prevention of disease progression and
complications. Tight glycemic control in type 1 diabetics, defined by an HbA1c value of <6.5%,
lowers the risk for the development and progression of microvascular disease. Currently, some
laboratories are reporting HbA1c values with an average glucose value estimate, calculated using
a formula. This approach permits the HbA1c result to be interpreted using the same units used
for daily glucose monitoring. Fructosamine, also known as glycosylated albumin, is used in some
institutions as a measure of glycemic control, because it reflects control over a shorter time frame
than the HbA1c level. Monitoring and managing dyslipidemia, using total cholesterol, low- and
high-density lipoprotein, and triglycerides as indicators, may lower the risk of developing macro-
Gestational diabetes vascular disease for both type 1 and type 2 diabetics. Trace excretion of urinary albumin, termed
represents any level “microalbuminuria,” is routinely measured in patients with DM as an early marker of nephropa-
of glucose intolerance thy. This test is not usually a part of routine urinalysis, and must therefore be ordered as a micro-
initially recognized albumin test, along with a creatinine level, on a random urine specimen (see Chapter 18).
during pregnancy, even if
it may have been present
but unrecognized before GESTATIONAL DIABETES MELLITUS
the pregnancy.
Description
Gestational DM represents any level of glucose intolerance initially recognized during pregnancy,
even if it may have been present but unrecognized before the pregnancy. When hyperglycemia
occurs for the first time during pregnancy, it usually develops late in the second or in the third
trimester. Most women will be normoglycemic after pregnancy. Among the complications of
untreated gestational DM are macrosomia (birth weight >4000 g or 8.82 lb), intrauterine fetal
demise, and pulmonary immaturity. Congenital malformations are increased only in women who
have preexisting diabetes, which may or may not have been clinically appreciated. Approximately
1 in 25 pregnancies in the United States is complicated by gestational DM, with a higher incidence
in some ethnic groups (up to 1 in 7 in Native Americans).
Diagnosis
Screening for gestational DM was previously recommended for all pregnant women. Some experts
suggest that screening of women at low risk for gestational DM is not cost-effective, although oth-
ers still advocate testing all pregnant women. These low-risk women are of Caucasian or Middle-
Eastern ancestry, less than 25 years old, of normal weight, have no first-degree relatives with DM,
and have no history of abnormal glucose metabolism. Women with a high risk for gestational DM
should be evaluated as soon as feasible into the pregnancy. All other women should be screened
between 24 and 28 weeks gestation, with the exception of women with clinical symptoms consis-
tent with gestational DM before 24 weeks, who should be tested when symptoms appear.
The ADA criteria for screening are shown in Table 17–6. Patients with an abnormal finding
with the screening OGTT (with 50 g glucose) must also have an abnormal confirmatory finding
with the OGTT (with 100 g glucose) to warrant the diagnosis of gestational DM. If a patient has
only 1 abnormal value during a confirmatory OGTT performed at 24 to 28 weeks of gestation,
some authorities recommend repeating the test at 32 weeks. The WHO currently advocates a
screening OGTT for gestational DM using a 75-g glucose bolus, which has been shown to be
more sensitive at detecting women at risk than the test using 50 g. However, the ADA chose not
to adopt this modification because of the wide use of the 50-g bolus.
TABLE 17–6 Laboratory Evaluation for Gestational Diabetes Mellitus (GDM)

Test Interpretation
1. Screening OGTT: 50-g glucose bolus PG (1 h post bolus):

• Patient need not fast • ≥140 mg/dL is abnormal
• <140 mg/dL is normal
2. Confirmatory OGTT: 100 g glucose Abnormal if at least 2 of the following 4 are met:
• Patient must fast • PG (prior to bolus): ≥95 mg/dL
• Only for patients with abnormal • PG (1 h post bolus): ≥180 mg/dL
screening OGTT • PG (2 h post bolus): ≥155 mg/dL
• PG (3 h post bolus): ≥140 mg/dL
3. Follow-up postpartum: fasting PG only Use criteria for nonpregnant adults in

• Only for patients who have had GDM; Table 17–2
to be performed at 6 months and every
3 years postpartum
OGTT, oral glucose tolerance test; PG, plasma glucose.
Six weeks after the end of a pregnancy complicated by gestational DM, the woman should be
retested. Normoglycemic women with a history of gestational DM should be rescreened at 3-year
intervals, and women with IFG or IGT should be screened more frequently.
HYPOGLYCEMIA The plasma glucose level

in hypoglycemic patients
Description decreases well below the
Hypoglycemia is a low plasma glucose state. Symptoms result from activation of the autonomic reference range, often
pathways and from inadequate glucose delivery to the central nervous system. This explains to less than 40 mg/dL.
the clinical features of hypoglycemia that are, in the acute form, intermittent episodes of sweat- Hypoglycemia is most
ing, tachycardia, anxiety, dizziness, slurred speech, double vision, and confusion, with complete commonly observed in
recovery on restoration of plasma glucose to normal levels. patients being treated for
diabetes.
The plasma glucose level in hypoglycemic patients decreases well below the reference range,
often to less than 40 mg/dL. Hypoglycemia can be divided into reactive postprandial hypoglyce-
mia and fasting hypoglycemia. Overall, hypoglycemia is most commonly observed in patients
being treated for diabetes.
• Reactive hypoglycemia: Reactive hypoglycemia may occur in patients who have had gastric
surgery, in children with inborn errors in enzymes leading to fructose intolerance, or
following ethanol consumption. Normally, the ingestion of a high-carbohydrate meal
increases the plasma glucose level and stimulates the release of an appropriate amount
of insulin. In reactive hypoglycemia, the peak concentration of insulin is inappropriately
high and causes the plasma glucose level to decrease below the reference range. Reactive
hypoglycemia is diagnosed if there are hypoglycemic symptoms, and a plasma glucose level
below 50 mg/dL following a high-carbohydrate meal, with resolution of symptoms after
administration of glucose.
• Fasting hypoglycemia: Fasting hypoglycemia may result from a variety of causes including
drugs (ethanol, sulfonamides, salicylate, and pentamidine), insulinoma and other islet
cell tumors, autoantibodies to insulin or its receptor, malignancy (such as sarcoma and
hematopoietic tumors), various inborn errors of metabolism, critical illness, and selected
endocrine disorders, among other causes. In general, fasting hypoglycemia can be classified
into hyperinsulinemic and nonhyperinsulinemic types. Patients who become hypoglycemic
as a result of excess insulin secretion from an insulinoma will have a decreased plasma
glucose level and/or an abnormal insulin to glucose ratio. Elevated blood insulin levels
independent of high-carbohydrate meals are found in patients with insulinomas. In addition,
patients with insulinomas have a high serum level of C-peptide, which is formed during
the conversion of proinsulin to insulin within the beta cells of the pancreas. C-peptide
appears in the blood in an approximately equimolar concentration with insulin and, thereby,
provides a reliable indication of insulin synthesis by the beta cells of the pancreas.
• Surreptitious insulin injection: Patients who inject themselves with insulin to produce
a hypoglycemic state will have the same hypoglycemic symptoms as patients with
hypoglycemia from other causes. However, these patients can be differentiated from
patients with insulinomas because, even though they have a high level of plasma insulin,
they have decreased levels of C-peptide. In insulin used for injection, the C-peptide
moiety is removed. The C-peptide is not present in patients with surreptitious insulin
injection because the insulin found in their blood is not synthesized from proinsulin in
their pancreas.
• Excess administration of sulfonylureas: As with insulin, patients who have purposely taken
sulfonylureas (an oral antidiabetic medication) in greater than prescribed doses suffer
from hypoglycemia. Because oral antidiabetic medications are not naturally occurring
compounds, a high serum concentration of these medications can reveal excess intake.
• Impaired liver function: Hypoglycemia can also occur in the presence of liver disease,
often when it is associated with excess alcohol intake or ingestion of certain medications.
Beta cell tumors are also Diagnosis

known as insulinomas
To diagnose hypoglycemia, the following 3 criteria (known as Whipple’s triad) must be met:
and are clinically
significant when they • Characteristic signs and symptoms of hypoglycemia
produce enough insulin • Blood glucose level below 45 to 50 mg/dL coincident with symptoms
to induce hypoglycemia. • Symptom reversal within 15 to 45 minutes of the administration of glucose, in the absence
(See the section “Islet Cell of cerebral edema
Tumors.”)
A portion of this chapter appeared previously in Clinical Laboratory Reviews (a publication for
the Massachusetts General Hospital physicians) 2000;8:2 and 1999;7:4. It has been included with
permission.
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C H A P T E R
The Kidney
William E. Winter 18
LEARNING OBJECTIVES
1. Recognize the association between the physiologic roles of the kidney
and the laboratory assays used to assess renal function.
2. Explain the basic concepts of the laboratory assays for renal function.
3. Understand the diagnosis of specific renal disorders using clinical
laboratory tests.
CHAPTER OUTLINE
Overview of Renal Disease 385 Patterns of Proteinuria 392
Clinical Laboratory Parameters 387 The Fractional Excretion of Sodium as an
Creatinine 387 Indicator of Tubular Function 392
The Glomerular Filtration Rate and Urinalysis 393
Creatinine Clearance 388 Stones and Crystals Found in Urine 395
Urea and the Blood Urea Nitrogen 389 Selected Additional Tests to Evaluate
The Blood Urea Nitrogen/Creatinine Renal Function 395
Ratio 390 Novel Biomarkers of Acute
Urine Protein Quantitation 390 Kidney Injury 396
OVERVIEW OF RENAL DISEASE

The homeostatic roles of the kidney include maintenance and regulation of fluid balance, acid/
base and electrolyte balance (eg, sodium, potassium, chloride, bicarbonate, calcium, phosphate,
and magnesium), conservation of glucose, amino acids, and proteins, the excretion of wastes,
and the production of hormones such as erythropoietin and 1,25-dihydroxyvitamin D. The renal
blood vessels provide blood to the glomerulus and the tubules for the generation of urine. The
glomerulus filters blood to create a plasma ultrafiltrate by retaining cells and proteins, whereas
the tubules “process” the plasma ultrafiltrate to urine, thereby concentrating wastes such as urea,
creatinine, nitrogenous wastes, and hydrogen ions.
Renal disease is suggested by any of the following findings:
1. Nonspecific symptoms of malaise, headache, visual disturbances, nausea, or vomiting
(eg, many of these findings suggest uremia or hypertension [see below]).
2. Flank pain (eg, from pyelonephritis), pain that radiates to the groin from the flank (eg, from
ureteral colic as a result of nephrolithiasis), or simple dysuria (eg, from a lower urinary
tract infection).
3. A reduction in the volume of urine output. In adults, oliguria, a pathologically reduced urine
output, is defined as less than 500 mL of urine produced per day. Anuria, which is essentially
absent urine production, is defined in adults as less than 100 mL of urine produced per day.
In infants, oliguria can be defined as urine output of less than 1 mL/kg/h, and in children
older than infants, oliguria is defined as urine output of less than 0.5 mL/kg/h.
385
386 CHAPTER 18 The Kidney
4. Hematuria, red blood cell casts, white blood cell casts, proteinuria, proteinaceous casts,
pyuria, or other abnormalities on urinalysis.
5. Discolored or malodorous urine (eg, from a urinary tract infection).
6. Elevations in the plasma or serum concentrations of creatinine or blood urea nitrogen
(BUN).
7. Malar rash (eg, from systemic lupus erythematosus).
8. Hypertension.
9. Otherwise unexplained hypokalemia or hyperkalemia, hypocalcemia, hypophosphatemia
or hyperphosphatemia, pathologic fractures, hypomagnesemia, acidosis, anemia, edema,
or bleeding.
Renal function should be evaluated when patients are taking drugs that can damage the kid-
ney (eg, gentamicin) or drugs whose metabolism and/or excretion is dependent on the kidney
(eg, low-molecular-weight heparin).
Nitrogen retention, as shown by an elevated BUN concentration, is termed “azotemia.” Azo-
temia can be classified as prerenal, renal, or postrenal. Prerenal azotemia refers to conditions with
reduced blood flow to the kidney, thereby reducing urine output and causing the retention of
waste products. Examples of prerenal causes of azotemia are congestive heart failure, GI hemor-
rhage, renal artery stenosis, and severe dehydration.
Renal azotemia indicates Renal azotemia indicates that the kidney itself is dysfunctional. The number of individual
that the kidney itself causes of intrinsic renal disease is large. In the broad view, however, renal azotemia results from
is dysfunctional. Renal diseases of the renal blood vessels, glomerulus, tubules, or interstitium. Glomerulonephridites
azotemia results may be the result of a primary process in the kidney (eg, autoantibodies against the phospho-
from diseases of the lipase A2 receptor causing membranous nephropathy) or a secondary disorder leading to glo-
renal blood vessels, merulonephritis (eg, anti-basement membrane autoantibodies causing Goodpasture syndrome).
glomerulus, tubules, A biopsy is often necessary to identify the type of glomerulonephritis (eg, lupus nephritis, acute
or mesangium. postinfectious [poststreptococcal] glomerulonephritis, IgA nephropathy, hereditary nephritis,
or rapidly progressive glomerulonephritis). The histopathologic characteristics of the different
glomerulonephridites and nephropathies are described in textbooks of anatomic pathology (eg,
podocyte effacement in minimal change, “tram-tracks” in membranoproliferative glomerulo-
nephritis, IgA deposition in IgA nephropathy, and “basket weaving” in Alport syndrome). It is
worth noting that IgA nephropathy can produce nephritis or nephrosis. Acute tubular necrosis
can cause renal failure. This may occur as a result of exposure to a toxin or as a result of ischemic
damage to the tubules.
Postrenal azotemia results from an anatomic obstruction to urine flow out of the kidney. The
ureter, bladder outlet, or urethra may be obstructed by a stone (eg, nephrolithiasis), congenital
anomaly, inflammatory lesion, or neoplasm.
Among prerenal, intrinsic renal, and postrenal-induced renal failure, the dominant etiology
is prerenal.
Uremia, unlike azotemia, is a clinical term that describes the patient’s signs and symptoms
when symptomatic end-stage renal failure is present. Findings in uremia include fatigue, head-
ache, restlessness, depression, altered sensorium, nausea, vomiting, diarrhea, hiccups, bleeding,
edema, shortness of breath, and pulmonary edema. Left untreated, uremia progresses to coma
and death. Renal failure produces a wide variety of adverse clinical and metabolic consequences
(Table 18–1).
Chronic renal failure is a deterioration in renal function that persists for more than 3 months.
It occurs with progressive renal damage, and is independent of the cause of kidney disease.
Chronic renal failure is the ultimate consequence of the loss of functioning nephrons. The domi-
nant etiologies of chronic renal failure in adults are multifactorial, as in patients with diabetes
mellitus, hypertension, glomerulonephritis, pyelonephritis/interstitial nephritis, cystic kidney
disease, and toxicity from drugs. A significant percentage of patients with chronic renal failure
have no known etiology for their disease.
Renal function can be assessed using a variety of clinical laboratory analyses. Acid/base and
electrolyte balance is initially assessed by ordering a profile of tests that includes sodium, potas-
sium, chloride, total serum CO2 (bicarbonate), BUN, creatinine, calcium, and glucose.
A more detailed analysis of acid/base balance would also include a measurement of arterial
blood gases (pH, pco2, po2, and calculated bicarbonate) and urine pH. The effect of erythropoietin
CHAPTER 18 The Kidney 387
TABLE 18–1 Selected Consequences of Untreated Renal Failure

Pathophysiology Immediate Consequences Later Possible Consequences
Salt and water retention Hypertension Heart failure, pulmonary edema
Potassium retention Hyperkalemia Cardiac arrhythmias
Phosphate retention Hypocalcemia Hyperparathyroidism with renal

Hyperphosphatemia osteodystrophy
Decreased synthesis of Hypocalcemia Hyperparathyroidism with renal

1,25-dihydroxyvitamin D osteodystrophy
Decreased production of Anemia Heart failure

erythropoietin
Decreased waste excretion Azotemia, acidosis Uremia
Decreased waste excretion Platelet dysfunction Bleeding tendency
is assessed by measuring the patient’s hemoglobin, hematocrit, and red blood cell indices.
The kidney’s role in producing active vitamin D, that is, 1,25-dihydroxyvitamin D, and controlling
phosphate excretion is evaluated, in part, through measurements of serum calcium and albumin
(or ionized calcium), phosphate, and parathyroid hormone (PTH). Measurements of 25-hydroxy
vitamin D assess vitamin D stores. 1,25-Dihydroxyvitamin D levels reflect the most active form of
vitamin D in the body. However, measurements of 1,25-dihydroxyvitamin D are rarely required.
Urinary tract infections are especially common in females. Females are more prone to uri-
nary tract infections than men because in females the urethra is shorter, and the distance from
the urethra to the anus is shorter than in males. Discussion of pathogenic organisms resulting in
bacterial infections of the kidney and urinary tract appears in Chapter 5.
CLINICAL LABORATORY PARAMETERS

Creatinine
Creatinine is a breakdown product of creatine and phosphocreatine, also known as creatine phos-
phate. Creatine is produced in skeletal muscle, the kidney, and the pancreas and is then trans-
ported to the tissues, especially the skeletal muscle and brain, via the bloodstream. Within cells,
creatine is phosphorylated to phosphocreatine via the enzymatic action of creatine kinase (CK).
Phosphocreatine provides a ready, rapid source of energy. For example, phosphocreatine is used
as a short-term energy source as required during a sprint.
With an approximate 1% to 2% daily turnover rate, creatine and phosphocreatine are metabo-
lized to creatinine at a fairly constant rate. Therefore, the plasma concentration of creatinine is usu-
ally stable day to day. Creatinine can be measured in the clinical laboratory by its ability to form
an orange-red-colored product in a chemical reaction with alkaline picric acid. This is the classic
Jaffe reaction. Creatinine can also be measured enzymatically using creatininase. Modern alkaline
picric acid methods have been improved to minimize interferences by other substances. Nonethe-
less, at this time creatinine measurements using the picric acid method can be falsely elevated by
a number of substances including ketones, glucose, and various drugs, such as cephalosporins and The glomerulus
sulfonamides. Using creatininase to measure creatinine, interferences are uncommon. is investigated by
Creatinine is freely filtered. However, 10% of the total excreted creatinine is secreted by the determining the GFR. GFR
tubules. Negligible amounts of creatinine are reabsorbed. The alkaline picric acid method over- is the number of milliliters
estimates serum creatinine by at least 10% because of endogenous positive interferences. Cre- of body fluid cleared by
atininase methods are calibrated to report a creatinine concentration comparable to creatinine the kidneys per unit time
measured by the alkaline picric acid method. Therefore, the creatininase methods used to mea- reported in mL/min.
sure serum or plasma creatinine also display a positive bias. Standardization of creatinine mea-
surements among analyzers has become an important goal for laboratory medicine.
The creatinine concentration in blood is inversely related to glomerular filtration rate
(GFR; see below). If the GFR declines by 50%, the plasma creatinine approximately doubles. The
creatinine concentration is directly related to skeletal muscle mass. Creatinine is higher in males
than in females, and increases with protein intake and with creatine intake. Creatine is sometimes
used as a “nutritional” supplement by body builders or athletes. The clearance of creatinine by the
kidney is a suitable estimate of GFR that is universally used by physicians. In the research labora-
tory, inulin clearance is an accurate method for measuring GFR. However, inulin clearance mea-
surements are not applicable to routine clinical medicine because inulin must be infused IV until
a constant concentration is achieved. Also in the research laboratory, renal plasma flow (RPF) can
be estimated by measuring para-aminohippurate (PAH) clearance. Both the RPF and GFR allow
the filtration fraction to be calculated (eg, the fraction of plasma that passes through the kidney
per unit time which crosses the glomerular capillary barrier to enter the Bowman space).
The Glomerular Filtration Rate and Creatinine Clearance

Laboratory evaluation of the kidney as discussed in this chapter centers on assessments of glo-
merular and tubular function. The glomerulus is investigated by determining the GFR. GFR is
the number of milliliters of body fluid cleared by the kidneys per unit time reported in mL/min.
Ideally, GFR is measured using a substance that is produced by the body at a constant rate that
is freely filtered by the glomerulus and is neither secreted nor reabsorbed by the tubules (ie, inu-
lin). As GFR is reduced, waste retention occurs. Measurable waste products excreted by the kid-
ney include creatinine, urea, and uric acid. With a decline in the GFR, these waste products are
retained and their circulating concentrations rise. Measurement of the GFR is a very important
assessment of renal function. A steady decline in GFR can serve as a harbinger of eventual end-
stage renal disease.
GFR measurements are most commonly based on the clearance of creatinine by the kidney.
This entails a serum creatinine measurement and a concurrent timed urine collection for the
measurement of excreted urinary creatinine and urine volume. In individuals aged 18 years and
above, an estimate of the GFR (eGFR) can be calculated solely from the serum creatinine and
various patient parameters such as age, sex, and ethnicity.
A complete urine collection is essential for an accurate determination of the creatinine clear-
ance because the equation contains a measurement of urine volume. The blood sample for serum
creatinine is usually collected at the beginning of the timed urine collection. The clearance is
expressed in terms of volume of fluid cleared per unit time (eg, mL/min).
The basic formula for creatinine clearance is as follows (serum creatinine and plasma creati-
nine are used interchangeably in the formulae):
Urine creatinine Urine volume (mL)

GFR measurements are ×
most commonly based Serum creatinine Collection time (minute)
on the clearance of
The clearance can be corrected for the patient’s body surface area to be compared with a standard
creatinine by the kidney.
body surface area of 1.73 m2.
This entails a serum
creatinine measurement
When corrected for body surface area, the formula for creatinine clearance is as follows:
and a concurrent timed Urine creatinine Urine volume (mL) 1.73
urine collection for the × ×
Serum creatinine Collection time (minute) Body surface area (m2)
measurement of excreted
urinary creatinine and For creatinine clearance (see the above formula), a 12- or 24-hour urine specimen is collected.
urine volume. The GFR in persons aged 18 years and above can be reliably estimated (also known as the
eGFR) solely from the patient’s serum creatinine, age, gender, and ethnicity (eg, African Ameri-
can or non-African American). The use of the modification of diet in renal disease (MDRD)
equation, which is:
eGFR = 186(SCr)−1.154 × (Age)−0.203 × F
where F = 0.742 for females and 1.210 for African Americans, provides GFR estimates compa-
rable to measured creatinine clearance when the GFR is less than 60 mL/min/1.73 m2. Because
of difficulties in obtaining a complete timed urine collection, the National Kidney Foundation
(NKF) advises that eGFR be used in place of creatinine clearance measurements when the GFR
is between 15 and 60 mL/min/1.73 m2. It is not presently advised that eGFR be calculated in chil-
dren because pediatric equations are not as well validated, unlike the MDRD equation that has
been well validated in adults.
TABLE 18–2 National Kidney Foundation Definitions of Kidney Damage Relative

to the Glomerular Filtration Rate (GFR)
Stage GFR Comment
0 ≥90 Normal kidney function and no proteinuria
1 ≥90 Kidney damage despite a normal or increased GFR
2 60-89 Mildly decreased GFR with evidence of kidney damage
3 30-59 Moderately decreased GFR

4 15-29 Severely decreased GFR
5 <15 Renal failure and dialysis or transplant needed

2
GFR is reported in mL/min/1.73 m .
The MDRD equation provides eGFR determinations that are reliable between 15 and
60 mL/min/1.73 m2. However, below 15 mL/min/1.73 m2 and above 60 mL/min/1.73 m2, GFR
should be estimated using the traditional creatinine clearance measurement. The lower limit of the
reference range for GFR is 90 mL/min/1.73 m2. However, the upper limit of the reportable eGFR
is 60 mL/min/1.73 m2. Therefore, there is a “gray” zone between the upper limit of the reportable
eGFR and the lower limit of the reference interval (90 mL/min/1.73 m2). Therefore, it is practical to
report eGFR values greater than 60 mL/min/1.73 m2 as simply “greater than 60 mL/min/1.73 m2”
with a comment that “the lower limit of the reference interval is 90 mL/min/1.73 m2.”
Many pathologic renal and systemic conditions can reduce the GFR. As GFR declines, cre-
atinine clearance can, however, begin to overestimate GFR. This is because the fraction of the cre-
atinine that is secreted by the tubules becomes a proportionately higher percentage of the urine
creatinine excreted as GFR declines. However, since clinical practice is almost always based on
assessment of the creatinine clearance, the difference between the “true” GFR and the creatinine
clearance as a reflection of the GFR is usually not problematic when making clinical judgments.
The development of renal impairment is frequently unrecognized until late in its course,
when intervention is less likely to be successful. Screening for reductions in GFR has recently
been championed by the NKF. The NKF provides guidelines for the interpretation of the GFR
result (Table 18–2). It defines kidney damage as any pathologic kidney abnormality reflected by
a marker of damage, as shown in a blood, urine, or imaging study. Kidney damage that is present
for more than 3 months is termed “chronic” kidney damage.
The NKF stresses that the creatinine clearance (unlike the eGFR) does provide useful infor-
mation in estimating the GFR in individuals who have exceptional dietary intakes (such as those
on vegetarian diets or those taking creatine supplements) or muscle mass changes (such as people
with amputations, malnutrition, or wasting conditions). Creatinine clearance measurements are
also valuable when deciding on the initiation of dialysis. This decision is made when the GFR is
<15 mL/min/1.73 m2; the eGFR is not reliable in this setting.
Urea and the Blood Urea Nitrogen

Urea is produced by the liver to create a metabolite of ammonia (nitrogen) that can be excreted in Modern laboratory
the urine. The nitrogen in ammonia is derived from the deamination of amino acids. methods actually
The initially developed laboratory measurements of urea depended on liberating nitrogen measure urea in serum
from urea in whole blood. Therefore, the term “BUN” was created. However, modern laboratory or plasma (and not in
methods actually measure urea in serum or plasma (and not whole blood) and back-calculate the the whole blood) with
nitrogen content, but the term “BUN” has persisted. back-calculation of the
Urea is freely filtered by the glomerulus. However, because ∼50% of urea is reabsorbed by the nitrogen content, yet the
tubules, urea clearance greatly underestimates GFR and, therefore, urea clearance is not usually term “BUN” has persisted.
determined. Furthermore, although creatinine production by the body occurs at a fairly constant
rate, providing stable serum creatinine concentrations over time (assuming there is no acute dis-
ease affecting the kidneys such as acute tubular necrosis), BUN levels can vary considerably. BUN
is affected by the patient’s state of hydration, protein intake, and the presence of large amounts of
TABLE 18–3 Clinical Use of the BUN/Creatinine (Cr) Ratio

Action
Both BUN and creatinine Do not calculate the BUN/Cr ratio

within the reference range
BUN and/or creatinine above the Calculate the BUN/Cr ratio (reference range: 10:1-20:1)
reference range • ≥20:1 suggests prerenal azotemia or early postrenal azotemia
• ≤10:1 suggests renal azotemia or late postrenal azotemia
blood in the gastrointestinal tract. If a large gastrointestinal hemorrhage occurs, the metabolism
of this additional protein originating from blood cells in the gastrointestinal tract leads to urea
production. With a decline in the GFR, BUN rises. Lastly, BUN can decline if there is liver failure,
leading to decreased urea production or when there is malnutrition and amino acids are “con-
served” for protein synthesis.
The 24-hour urea excretion in the urine can be used as an assessment of nutritional replace-
ment in malnourished patients. If sufficient nitrogen is present in the diet and utilized by the
body, normal levels of urea are excreted in the urine.
The Blood Urea Nitrogen/Creatinine Ratio

If either the creatinine or BUN concentrations are above the upper limit of the reference interval,
it is advised that the BUN to creatinine ratio (BUN/Cr) be calculated (Table 18–3). The normal
BUN/Cr ratio is between 10:1 and 20:1. The ratio is helpful in determining the cause of renal
impairment.
If the BUN/Cr ratio is 20:1 or higher, prerenal azotemia is likely to be present. Prerenal azo-
temia results from a reduction in the GFR while the kidney tubules are functioning.
The explanation why the BUN rises to a greater extent than creatinine involves 2 obser-
vations: 1) renal tubular secretion of creatinine persists even as GFR declines, opposing what
would otherwise be a rise in serum creatinine, and 2) with decreased renal blood flow, the rate of
capillary blood flow around the renal tubules is reduced, providing more time for the reabsorp-
tion of urea out of the urine in the tubules and back into the circulation, raising the serum BUN
concentration.
When the BUN/Cr ratio is near 10:1 and creatinine and/or BUN are elevated, renal azotemia
is likely, assuming that a chronic urinary tract obstruction has been excluded. In cases of renal
azotemia, the BUN and creatinine rise proportionate to one another because, in part, tubular
dysfunction will not maintain the tubular secretion of creatinine.
In the early phase of postrenal obstruction, the BUN/Cr ratio is ∼20:1 because urea is reab-
sorbed from urine that is “stagnant” in the excretory system because of the anatomic obstruction.
Therefore, if the BUN/Cr ratio is elevated at the time of patient presentation, the treating physi-
cian is obligated to consider the possibility of an anatomic obstruction to urine flow, as well as
prerenal azotemia. If there is persistent urinary tract obstruction, postrenal azotemia can evolve
into renal azotemia from damage to the kidneys. Obstruction appears to trigger inflammation in
the glomerulus. If renal impairment then supervenes, the BUN/Cr ratio would fall to 10:1, similar
In adults, proteinuria to other conditions associated with intrinsic renal disease.
greater than 1 g per day
is considered to be very
significant clinically. Urine Protein Quantitation
Levels of protein The general health of the kidney is assessed in part by the measurement of urinary protein excre-
excretion of 3.5 g per day tion. In normal adults, 24-hour urinary protein excretion does not exceed 150 mg. If one assumes
or greater are consistent that a normal adult urine output is 1500 mL per day and a maximum of 150 mg of protein is
with nephrosis. excreted per day, the urine protein concentration should not exceed approximately 10 mg/dL.
Elevated concentrations of protein in the urine can result from glomerular disease, tubular dis-
ease, overflow from elevated concentrations of plasma proteins, such as immunoglobulins or
immunoglobulin light chains in patients with myeloma, urinary tract inflammation, as found in
interstitial nephritis or urinary tract infection, trauma, or neoplasia.
TABLE 18–4 Interpretation of Albumin Excretion in the Urine

Units Normal Microalbuminuria Clinical Albuminuria
Spot collection μg/mg Cr <30 30-299 ≥300
Timed urine μg/min <20 20-199 ≥200
24-Hour urine mg/24 h <30 30-299 ≥300

Cr, creatinine.
In adults, proteinuria greater than 1 g per day is considered to be very significant clinically.
Levels of protein excretion of 3.5 g per day or greater are consistent with nephrosis. Nephrosis
is the clinical syndrome of massive proteinuria, hypoalbuminemia, edema, and hyperlipidemia.
Primary renal diseases causing nephrosis include minimal-change disease, focal segmental glo-
merulosclerosis, membranous nephropathy, membranoproliferative glomerulonephritis, and IgA
nephropathy. Secondary causes of nephrosis include diabetes mellitus, amyloidosis, lupus, drugs
(eg, gold, penicillamine, heroin), infections (eg, malaria, syphilis, HBV, HIV), and malignancy
(eg, carcinoma, melanoma).
In children, an elevated level of urinary protein excretion is >4 mg/m2/h. Nephrotic range
proteinuria in children can be defined as >40 mg/m2/h.
The most cost-effective way to initially screen for proteinuria is urine protein dipstick test-
ing. In this semiquantitative system, proteinuria is reported as negative, trace (10-20 mg/dL),
1+ (30 mg/dL), 2+ (100 mg/dL), 3+ (300 mg/dL), or 4+ (1000-2000 mg/dL). Urine dipsticks for
protein measurements are relatively insensitive to immunoglobulin light chains and, thus, a nega-
tive dipstick does not exclude Bence Jones (monoclonal light chain) proteinuria. A more accurate
measure of proteinuria can be made using a 24-hour urine sample. The urine protein concentra-
tion in milligrams per deciliter is multiplied by the urine volume in milliliters per 24 hours yield-
ing milligrams of protein excreted per 24 hours.
Minimal but persistent amounts of albumin excretion in the urine (eg, microalbuminuria) Minimal but persistent
are associated with diabetic nephropathy and with hypertensive renal damage. For this reason, amounts of albumin in the
people with diabetes mellitus are screened for minimal albumin excretion, also known as micro- urine are associated with
albuminuria. The albumin measurement is carried out using an immunoassay to provide analyti- diabetic nephropathy and
cal sensitivity, accuracy, and reproducibility. Microalbuminuria can be reported as the albumin with hypertensive renal
to creatinine ratio obtained on a random urine sample, the albumin excretion in milligrams per damage. For this reason,
minute on a timed urine sample collection (eg, a 4-, 6-, or 12-hour collection), or the albumin people with diabetes
excretion per 24 hours when a 24-hour urine sample is collected. Table 18–4 provides an inter- mellitus are screened
for minimal albumin
pretation of microalbumin results.
excretion, also known as
It is recommended that all patients with type 2 diabetes mellitus be tested yearly for micro-
microalbuminuria.
albuminuria from the time of diagnosis. For type 1 diabetes mellitus patients, testing is recom-
mended to be performed annually beginning 5 years after the diagnosis of diabetes mellitus.
Screening can begin with protein dipstick testing. If the dipstick is positive, microalbumin testing
can be bypassed and testing should proceed to a 24-hour urine collection for the measurement
of urine protein excretion. For patients with a negative dipstick result for proteinuria, microalbu-
minuria testing can be performed. If microalbuminuria is detected, a second sample should be
obtained within 3 months. If the second sample does not display microalbuminuria, a third, “tie-
breaker” sample is obtained. Thus, microalbuminuria must be present in 2 of 2 or in 2 of 3 samples
to classify the patient as having persistent microalbuminuria.
With persistent microalbuminuria, the diabetic patient is diagnosed with stage 3 (eg, incipi-
ent) diabetic nephropathy (Table 18–5). Stage 1 nephropathy immediately follows the diagnosis
of type 1 diabetes mellitus and is characterized by renal hypertrophy and hyperfiltration. These
patients have an elevated GFR from the expanded plasma volume induced by hyperosmolality
caused by hyperglycemia. With the initiation of insulin treatment, stage 1 resolves but clinically
silent histologic changes subsequently occur in the glomerulus with mesangial hypertrophy and
thickening of the glomerular basement membrane as observed on electron microscopy. This
is stage 2 diabetic nephropathy. With the recognition of incipient nephropathy (stage 3) and
intervention with improved glycemic control and the administration of antihypertensive drugs
TABLE 18–5 Laboratory and Blood Pressure Findings in Diabetic Nephropathy

Stage GFR UAE Dipstick Proteinuria Blood Pressure
1 Increased Normal Transient Normal
2 Normal Normal Negative Normal
3 Normal Increased Negative ± Increased
4 Decreased Increased Positive Increased

5 Severely decreased Increased Positive Severely increased
GFR, glomerular filtration rate; UAE, urinary albumin excretion.
(eg, angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers), further pro-
gression to frank diabetic nephropathy can be averted or at least delayed. Proteinuria by dip-
stick, a falling GFR, and persistent hypertension identify stage 4 diabetic nephropathy. Stage 5
nephropathy is characterized by the development of end-stage renal failure eventually requiring
either dialysis or transplantation.
Patterns of Proteinuria
When proteinuria is diagnosed, the subsequent diagnostic issues concern the cause of the pro-
teinuria and the structural portion of the kidney that is functionally impaired. The glomeru-
lus normally retains all plasma proteins with a molecular weight of greater than ∼100,000 Da
(Table 18–6). Variable amounts of plasma proteins with molecular weights between ∼10,000
and ∼100,000 Da are excreted into the urine. This includes albumin with a molecular weight of
∼69,000 Da and free immunoglobulin light chains with a molecular weight of ∼25,000 Da. Plasma
proteins below ∼10,000 Da, such as insulin, are essentially freely filtered by the kidney.
Urine protein electrophoresis can identify the following patterns of protein loss: glomerular,
tubular, overflow, and nonselective proteinuria. Glomerular proteinuria is characterized by albu-
minuria and the excretion of beta globulins, notably transferrin. Tubular proteinuria is recognized
in urine protein electrophoresis by an alpha-2 doublet in addition to increased albumin excretion.
Overflow proteinuria can result from a monoclonal immunoglobulin in high concentration
in the plasma that “spills over” into the urine. For example, free monoclonal antibody light chains
are detected by urine protein electrophoresis as an “M-spike,” a band of restricted mobility, and
confirmed to be present by immunofixation electrophoresis (IFE). The light chain loss occurs
because the ability of the tubules to reabsorb filtered protein is exceeded at high levels of pro-
teinuria. With extensive renal injury, intact monoclonal immunoglobulins can be lost. Persis-
tent glomerular proteinuria can injure the tubule, later resulting in a combined glomerular and
tubular proteinuria. The excretion of multiple low-molecular-weight proteins that arise as part
of the inflammatory acute-phase response can also produce overflow proteinuria. Nonselective
proteinuria, which can occur with severe renal dysfunction, is identified when the urine protein
electrophoresis pattern is similar to that of serum.
The Fractional Excretion of Sodium as an Indicator

of Tubular Function
A readily available test of Tubular dysfunction can result in many abnormalities: glycosuria, amino aciduria, renal tubu-
the resorptive function lar acidosis (bicarbonate wasting or failure to generate new bicarbonate), electrolyte wasting
of the tubules is the (eg, hyponatremia, hypokalemia, and hypophosphatemia), and tubular proteinuria (see above).
“fractional excretion of A readily available test of the resorptive function of the tubules is the “fractional excretion of
sodium (FENa).” FENa sodium (FENa).”
is calculated using FENa is calculated using creatinine and sodium measurements in serum or plasma and a
creatinine and sodium simultaneously collected spot urine. The equation for the FENa is given as follows:
measurements in
serum or plasma and a [UNa+] [SCr]
simultaneously collected FENa = × 100
[SNa+] [UCr]
spot urine.
TABLE 18–6 Approximate Molecular Weights of Selected Plasma Proteins

Location on Serum Protein Electrophoresis Gel Approximate Molecular Weight (kDa)
Prealbumin zone
Retinol-binding protein 21
Transthyretin 54
Albumin zone
Albumin 69
Alpha-1 globulin zone
Alpha-1 antitrypsin 54
High-density lipoprotein 200-400
Thyroxine-binding globulin 54
Alpha-1-acid glycoprotein 40
Prothrombin 72
Alpha fetoprotein 69
Alpha-2 globulin zone
Alpha-2 macroglobulin 800

Haptoglobin 86
Ceruloplasmin 160
Antithrombin 58
Erythropoietin 38
Beta globulin zone
Transferrin 77
C-reactive protein 115-140
Complement component 3 185
Beta2-microglobulin 12
IgA 170
Gamma globulin zone
IgM 900
IgG 160
Also see serum protein electrophoresis in Chapters 2 and 3.
The unit is percent sodium excreted (%). Normally the FENa is less than 1%. If there is tubular dis-
ease or injury, such as acute tubular necrosis, sodium wasting will occur and the FENa can exceed
1%. Tubular reabsorption of sodium is 100% minus the FENa. FENa calculations are not valid
when patients are treated with diuretics because the diuretic will induce urinary sodium loss. Examination of the
physical, chemical, and
microscopic contents
Urinalysis of urine constitutes
Examination of the physical, chemical, and microscopic contents of urine constitutes urinalysis urinalysis testing.
testing. The physical characteristics of the urine include its color, clarity, and specific gravity. A urinalysis should
Chemical analyses of urine include pH and detection of glucose, protein, blood, ketones, biliru- complement BUN and
bin, urobilinogen, nitrite, and leukocyte esterase. The microscopic examination is an assessment creatinine testing in any
for cells, bacteria, crystals, casts, lipids, and contaminants. patient undergoing a
If the urine dipstick is completely normal, some laboratories will not perform the micro- renal evaluation.
scopic examination. A urinalysis should complement BUN and creatinine testing in any patient
undergoing a renal evaluation.
The clinical significance of positive findings in urinalysis studies is briefly detailed below.
Normal urine is Normal urine is straw-colored. With dehydration, the color intensifies. With higher levels
straw-colored. With of fluid intake, the straw color is less intense. The color of normal urine is produced largely by
dehydration, the color pigments present in the diet, such as the pigments in vegetables, as well as metabolites of bile.
intensifies. With higher Patients with elevated urine bilirubin or urobilinogen can have urine that is darkly colored, or
levels of fluid intake, the some patients produce green urine because of the oxidation of bilirubin to biliverdin.
straw color is less intense. The dipstick for blood reacts with heme. Heme is detected whenever red blood cells,
The color of normal urine hemoglobin, and/or myoglobin are present in urine. Therefore, dipstick positivity for blood
is produced largely by does not identify whether red blood cells, hemoglobin, or myoglobin are present singly or in
pigments present in the
combination.
diet, such as the pigments
Hematuria refers to blood in the urine. It may be plainly visible (gross hematuria) or red
in vegetables, as well as
metabolites of bile.
blood cells may only be visible microscopically (microscopic hematuria). On a fresh urine sample,
microscopic examination can reveal the presence of red blood cells and/or red blood cell casts. If
red blood cell casts are present, nephritis or severe tubular injury is likely. Red blood cells in the
absence of casts indicate bleeding into the urinary tract or the presence of a hemorrhagic coagu-
lopathy. Casts are fragile, and therefore they are most likely to be found in a fresh, early morning
urine specimen. If red blood cells are present in the urine but sample analysis is delayed, casts
can fall apart and cells can lyse. Therefore, immediate analysis of a recently collected urine is best.
In a fresh urine sample lacking red blood cells but tests positive for blood, either hemoglobin
or myoglobin is present. Free hemoglobin can enter the urine in cases of intravascular hemolysis.
Myoglobin is released with muscle disease. Trauma can release large amounts of myoglobin into
the blood. Both hemoglobin and myoglobin are toxic to the renal tubules.
Hemoglobin and myoglobin can be distinguished by precipitation methods and via centrifu-
gation. In the precipitation methods, saturated ammonium sulfate is added, which precipitates
hemoglobin but not myoglobin. Therefore, after precipitation if the dipstick for blood is positive,
myoglobin is present. In centrifugation methods, the urine sample is centrifuged after application
over a filter that retains hemoglobin but permits myoglobin to enter the infranatant. If the infra-
natant is dipstick positive for blood, myoglobin is present.
Proteinuria is thoroughly described earlier in this chapter.
Pyuria refers to increased white blood cells in the microscopic urine sediment. This is often
considered to be at least 5 white blood cells per high-powered field. A test for leukocyte esterase
enzyme activity, found in neutrophils, is on most urinalysis strips and can detect this activity
whether the neutrophil is intact or disrupted. White blood cell casts originate in the tubules simi-
lar to red blood cell casts. White blood cell casts are consistent with pyelonephritis or noninfec-
tious interstitial inflammation.
Urinary casts are formed in the kidney tubules and are indicators of renal disease. Cellu-
lar casts can be formed by red blood cells, white blood cells, or renal tubular (epithelial) cells.
Granular casts, which do not contain intact cells, and waxy casts (both derived from degenerating
tubular cells) can also be found in patients with kidney disease. Hyaline casts are composed of
protein. They can be observed in the absence of disease.
Bacteriuria may be detected by a nitrite test on a urinalysis reagent strip, which is sensitive
to the presence of clinically significant urinary bacteria concentrations. However, not all bac-
teria convert nitrates to nitrites. Also the urine must be retained in the bladder for some hours
(approximately 4 hours or more) for this conversion to occur. Bacteriuria is often asymptomatic
but a positive test result may reflect bacterial infection nonetheless. It is frequently accompanied
by pyuria.
Urine glucose is not useful to diagnose or monitor patients with diabetes mellitus. There is
only an approximate association between the level of plasma glucose and urinary glucose, as the
renal threshold for glucose varies considerably among different individuals. This being said, if
glycosuria is detected on a routine urinalysis, diabetes mellitus should be considered as a possible
explanation. Trace glycosuria can be observed in normal pregnancies in the absence of diabetes
mellitus because there is a reduction in the tubular threshold for the reabsorption of glucose dur-
ing pregnancy.
Urine pH can be altered by conditions associated with metabolic acidosis or alkalosis. Freshly
collected urine specimens should have a pH between 5.0 and 6.5. A urine pH greater than 8.0 sug-
gests delayed analysis or bacterial contamination. On standing in an open container, CO2 leaves
the urine raising the pH. Bacteria can cleave urea to ammonia (NH3) and CO2. When ammonia
reacts with water, ammonium ion (NH4+) and hydroxyl ion (OH-) are formed. Increased hydroxyl
ion raises the pH.
Urine specific gravity is defined as the ratio of the weight of urine to the weight of an equal
volume of water. It provides an assessment of the capacity of renal tubules to concentrate or to
dilute urine. The specific gravity of urine should range between 1.003 and 1.035.
Bilirubin should not be present in the urine, and when it is detected, it is indicative of liver
dysfunction or biliary obstruction. Bilirubin present in the urine is conjugated, water-soluble
bilirubin.
Urinary urobilinogen is derived from bilirubin that is degraded by bacteria in the gastro-
intestinal tract. Urobilinogen then undergoes enterohepatic recirculation to then be excreted in
the urine. Conditions in which there is an elevated urinary urobilinogen include liver disease,
because of failure to remove the urobilinogen from the blood, and hemolytic anemia in which
bilirubin production increases the generation of urobilinogen.
Ketones most commonly appear in the urine of patients who have poorly controlled diabetes If there is a sufficient
mellitus, although they can also appear in stressed hospitalized patients who do not have diabetes amount of stone
mellitus and in fasting patients. material for analysis,
the composition of the
stone can be determined.
Stones and Crystals Found in Urine The value of knowing
The process of kidney stone formation is known as nephrolithiasis or urolithiasis. The presence of the composition of the
a kidney stone is often associated with severe pain radiating from the back and/or flank into the stone is that information
groin. Although most stones pass spontaneously, some do not. The size of the stone, among other may be derived about the
contributing factors to its
factors, determines whether the stone will be passed or retained. Stones can form when there is
formation.
increased excretion of the components found in stones or the urinary volume is decreased, lead-
ing to elevated concentrations of urinary components. Calcium, phosphate, and oxalate are the
most commonly found chemical constituents in renal stones, and less commonly identified are
urate and cystine.
If there is a sufficient amount of stone material for analysis, the composition of the stone
can be determined. The value of knowing the composition of the stone is that information may
be derived about the contributing factors to its formation. This can lead to treatment, sometimes
involving dietary modification.
An elevated concentration of calcium in the urine can lead to the generation of calcium
oxalate and calcium phosphate stones. An increased concentration of oxalate in the urine can
occur in patients who have an excess absorption of dietary oxalate. In Crohn disease, there is
increased absorption of oxalate from the ileum. Cystine can accumulate in the urine when there
is defective transport of cystine out of the urine by the proximal tubules, allowing cystine to reach
a concentration at which it becomes insoluble in the urine. High concentrations of urinary uric
acid are found in patients with gout, and such patients are predisposed to form urate stones, par-
ticularly when the urine has a pH below 5.4. Stones consisting of calcium carbonate and struvite
(MgNH4PO4) can occur in patients with urinary tract infections, particularly those caused by
Proteus. Many patients presenting with a kidney stone have no identifiable underlying cause for
its formation.
Crystals are frequently observed in a microscopic urine examination, and the majority are
normal urinary components. However, in patients predisposed to forming kidney stones, the
crystals may provide information that suggests the composition of the stone.
Selected Additional Tests to Evaluate Renal Function Many patients presenting

with a kidney stone have
Cystatin C no identifiable underlying
Cystatin C is a low-molecular-weight protein of ∼13,000 Da that is produced by the body at a con- cause for its formation.
stant rate. Because cystatin C appears to be cleared solely by the kidney, elevated cystatin C levels
are inversely proportional to the GFR. Epidemiologic data demonstrate that increased cystatin
C levels are positively correlated with mortality. Because creatinine measurements are readily
available (and inexpensive), cystatin C is unlikely to replace creatinine clearance measurements
in the near term.
Uric Acid
While it is true that uric acid concentrations rise as the GFR declines, uric acid is not a very
helpful indicator of GFR. This is because serum uric acid levels vary widely according to diet.
High-protein diets elevate uric acid, as does high cellular turnover. Neoplasias with high cellular
turnover rates elevate uric acid as does cell death from chemotherapy. Uric acid levels are mark-
edly affected by variation in the rates of production and reabsorption of uric acid, as found in
patients with gout.
Calcium, Phosphate, and Parathyroid Hormone

Calcium, phosphate, and bone metabolism is impaired in patients with renal failure. PTH normally
stimulates a net decrease in calcium excretion in the urine, as it stimulates calcium reabsorption
in the distal tubule. PTH increases phosphate excretion into the urine, as it decreases the loss of
calcium into urine. See Chapter 22 for additional information on this topic.
Novel Biomarkers of Acute Kidney Injury

Presently a major goal of nephrology is to identify an early marker of acute kidney injury that will
predict the development of acute renal failure. Such marker(s) would be analogous to markers
of myocardial necrosis (eg, cardiac troponin T or cardiac troponin I). Many markers are under
investigation including lipocalins (eg, neutrophil gelatinase-associated lipocalin-2; NGAL), heat
shock proteins (eg, HSP72), interleukins (eg, IL-18), and a variety of other proteins (eg, kidney
injury molecule-1, cystatin C (as noted above), N-acetyl-β-d-glucosaminidase, and liver fatty
acid-binding protein). One diagnostics company offers an assay for urinary NGAL on their auto-
mated immunoassay platform. Presently, none of these markers are used routinely and their diag-
nostic and prognostic values are under investigation.
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C H A P T E R
Male Genital Tract

Mark H. Wener, Charles H. Muller, and D. Robert Dufour 19
LEARNING OBJECTIVES
1. Understand how prostate-specific antigen (PSA) is used in the monitoring of
prostate cancer, and controversies regarding use of PSA as a cancer screening
test.
2. Learn how β-human chorionic gonadotropin (β-hCG), alpha-fetoprotein
(AFP) and lactate dehydrogenase (LD) levels are used in the management of
patients with certain germ cell testicular tumors.
3. Learn how tests of androgen metabolism and regulation can be used in
diagnosis of male gonadal dysfunction.
4. Learn the major causes of male infertility, and the major tests involved in
semen analysis.
CHAPTER OUTLINE
Introduction 397 Testicular Cancer 399
Prostate Cancer 397 Male Gonadal Dysfunction 400
Prostate-specific Antigen 398 Male Infertility 401
INTRODUCTION
The penis, testes, epididymis, vas deferens, seminal vesicles, and the prostate comprise the male
genital tract. Circulating markers have been identified for prostate cancer and testicular cancer.
For that reason, a discussion of these tumors and their serum markers is presented in this chapter
(Table 19–1). Also, laboratory tests are often used in evaluating men with gonadal dysfunction
and men who may be subfertile, infertile, or sterile. A summary of these tests and their usage is
also provided. The male genital tract is the site of many infectious diseases, a significant propor-
tion of which are sexually transmitted. These are discussed in Chapter 5.
PROSTATE CANCER
Description
Prostate cancer is a common malignancy of men that increases in incidence with age. It is
second only to nonmelanoma skin cancer as the most commonly diagnosed cancer in men (over
150 cases/100,000 men), and second only to lung cancer as the most common cause of cancer
death in males. However, most cases are slowly progressive and do not cause major morbidity
or lead to death. A major unresolved challenge is differentiating the rapidly progressive and fatal
form of prostate cancer from the indolent forms that do not cause death. Mortality associated with
the disease has been decreasing. This has been attributed by some to early detection, although
397
398 CHAPTER 19 Male Genital Tract
TABLE 19–1 Clinical Utility of Serum Tumor Markers for Prostate Cancer
and Testicular Cancer
Prostate Cancer: Testicular Germ Cell Tumors
Cancer Purpose Prostate-specific Antigen (PSA) (LD, AFP, hCG)
Screening Controversial for men older than Not useful

50 years
Establishing a diagnosis Not useful Can suggest histologic type(s) present,

especially for small clusters of 1 tumor
type that may be missed by histology
Indicator of disease extent If PSA <20 ng/mL, bone Of use in identifying clinically
metastasis unlikely undetectable metastatic disease
Monitoring response Useful to monitor success Useful; markers should fall to

to treatment of treatment undetectable with successful treatment
Monitoring for recurrence Useful Useful
a systematic review of published studies has shown no consistent difference in prostate cancer
mortality between those who have and those who have not been screened for the disease. The
use of laboratory assays to measure the serum prostate-specific antigen (PSA) concentration,
however, has had the greatest impact on increased detection of this cancer.
Prostate-specific Antigen
PSA is a serine protease enzyme (also called human kallikrein 3) synthesized almost exclusively
by the prostate and secreted into the seminal fluid. A small amount is also found in the blood. In
the blood, PSA is largely bound to enzyme inhibitor proteins such as alpha1-antichymotrypsin
and alpha2-macroglobulin. A small fraction of circulating PSA is free (unbound).
PSA levels in blood generally correlate with the size of the prostate. The larger the gland,
the higher the PSA value. PSA may also increase transiently after a vigorous rectal examination,
and after prostate biopsy or surgery. Inflammation and infarction of the prostate can also cause
increased PSA, which returns to normal gradually. It is therefore recommended that elevated PSA
levels should be confirmed by repeat measurement (at least 2-3 months apart) before any other
action is taken, to exclude 1 of these insignificant causes of high PSA.
Prostate disease is common in men after the age of 50 years, and by age 70 years the majority
of men have prostate disease. The 2 major diseases of the aging prostate are prostate carcinoma
and benign prostatic hyperplasia (BPH). A number of factors have been evaluated to try to distin-
guish between these causes of increased prostate size and/or increased PSA levels. Both prostate
cancer and BPH contribute to elevations in serum PSA.
In most laboratories, the PSA threshold considered positive for cancer screening is >4 ng/mL;
above that threshold concentration, the positive predictive value for prostate cancer (the likeli-
hood that cancer will be found in a biopsied prostate gland) is about 30%. In general, PSA is
PSA has been used for increased to a greater extent in prostate cancer than in BPH; PSA is rarely >20 ng/mL in BPH, and
several purposes related in only about 10% of cases is it >10 ng/mL, so higher values suggest cancer. A high PSA in a man
to prostate cancer:
with a small prostate gland on rectal examination is more worrisome for cancer than a similar
screening (testing
PSA value in a person with a very large gland. The ratio of free PSA/total PSA may be a better
in persons without
diagnostic marker for prostate cancer than total PSA; in general, a lower proportion of free PSA is
symptoms or signs of
disease), prediction found in patients with prostate cancer, but there is a wide overlap in values.
of the course of disease, PSA has been used for several purposes related to prostate cancer: screening (testing in per-
prediction of the stage of sons without symptoms or signs of disease), prediction of the course of disease, prediction of the
disease, and follow-up stage of disease, and follow-up after treatment. The most controversial use of PSA measurements
after treatment. The is in screening. Two large randomized trials have been published with long-term outcomes of
most controversial use prostate cancer screening. The US Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial
of PSA measurements is showed no effect on cancer-specific or all-cause mortality after 10 years. The European Random-
in screening. ized Study of Screening for Prostate Cancer found that screening was associated with reduced
CHAPTER 19 Male Genital Tract 399
cancer-specific mortality in men aged 55 to 69 years after 9 years of follow-up; to prevent 1 cancer
death, 1400 men would need to be screened and 48 treated.
Because of the side effects of diagnostic biopsy and complications of treatment of prostate
cancer and the minimal, if any, survival benefit from prostate cancer screening, in 2012, the
US Preventive Services Task Force recommended that men not be screened for prostate cancer.
Guidelines for prostate cancer screening from other professional groups, including the American
Urological Association (AUA) and the American College of Physicians (ACP), recommend indi-
vidual decision making with information provided to the patient about both potential risks and
benefits, and that screening might be reasonably offered to men between ages 50 (for the ACP) or
55 (for the AUA) and 69.
It should be noted that PSA is not highly sensitive for detecting cancer. It is estimated that
only about 50% to 60% of those with localized and potentially curable cancer have increased PSA,
and recent studies have found that many patients with less well-differentiated prostate cancers
actually have PSA values as low as 1 ng/mL.
Limited evidence suggests that the rate of rise in PSA can predict more aggressive cancers.
A review of published studies found that men with more rapid rises in PSA (a rise >0.35 ng/mL
per year, 10 years before a definitive diagnosis, or a rise >2 ng/mL in the year before a definitive
diagnosis) were far more likely to have recurrence after surgery and to die from cancer than those
with more slowly rising PSA. In men who have decided to not have surgery, the rate of rise of PSA
was also found to be predictive; if the PSA doubling time was less than 3 years, the likelihood of
locally progressive disease was high, while it was very low for those whose PSA increased less
than 2-fold over 10 years. More studies will be needed to confirm these findings and to determine
whether this information can be useful in determining treatment.
PSA measurement is of some use in the initial staging of a patient with prostate cancer. In
general, the higher the PSA, the less likely that cancer is localized to the prostate and the more
likely that it has spread. Distant metastases are rare in persons with PSA <20 ng/mL, so perfor-
mance of imaging studies of bone (the most common site of metastasis) for preoperative staging
of cancer has little benefit in those with lower PSA values.
The most widely accepted use of PSA is to monitor patients after treatment. Since about 99%
of prostate cancers produce PSA, and since PSA is made almost solely in the prostate, successful
surgical removal of the gland (and cancer) should result in a serum PSA less than 0.1 ng/mL by
3 months after surgery. Failure of PSA to become undetectable indicates residual cancer that was
not removed by surgery. With recurrence of cancer, PSA levels increase up to a year and a half
before clinical evidence of recurrent cancer, allowing treatment of persons with rising PSA before
their clinical condition deteriorates. With radiation therapy, PSA typically will fall to normal
(usually to <1 ng/mL by 1 year after completion of radiation) with successful treatment, but will
usually not be undetectable. Because prostate cancer responds to androgens, removal of the testes
and the use of drugs that block androgen production are widely used to treat metastatic prostate
cancer. The production of PSA is androgen dependent. Rarely, PSA levels will fall dramatically
with androgen deprivation even though there is little or no change in the amount of tumor. In
most cases, though, PSA is a reliable marker of tumor response to androgen deprivation as a treat-
ment for prostate cancer.
More than 90% of

TESTICULAR CANCER testicular cancers arise
Description from germ cell tumors.
Germ cell tumors often
The 2 major categories of testicular tumors are germ cell tumors (which include seminomas and
produce substances that
nonseminomatous germ cell tumors [NSGCT]) and sex cord or stromal tumors (mainly Ley- can be used as tumor
dig cell and Sertoli cell tumors). Seminomas and NSGCT comprise more than 90% of testicular markers to evaluate the
cancers. Most persons with germ cell tumors have a mixture of histologic varieties. Testicular patient for complete
germ cell tumors have a peak incidence in 15- to 34-year-old males, and are the most common removal of the tumor,
type of tumor found in men of that age group. Testicular cancer is most commonly identified detect recurrent cancer,
by finding an enlarged testicle during a routine physical or by a man on self-examination. If and monitor treatment for
an ultrasound examination confirms the presence of an intratesticular mass, surgery is usually any residual or recurrent
performed quickly to remove the testicle, its adnexa, and a long segment of the spermatic cord tumor.
(radical orchiectomy). The diagnosis of testicular cancer, like most other cancers, is made by his-
topathologic examination of the testicle.
Diagnosis
Germ cell tumors often produce substances that can be used as tumor markers to evaluate the
patient for complete removal of the tumor, detect recurrent cancer, and monitor treatment for
any residual or recurrent tumor. The 3 important serum tumor markers for testicular cancers
are human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), and lactate dehydroge-
nase (LD). LD elevation, while present in about 50% of seminomas and 10% of NSGCT, is not
specific, since damage to muscle, liver, cardiac, and other tissue damage can raise LD levels in
the blood. Elevation of the LD-1 isoenzyme is characteristic of testicular tumors, but LD-1 is
also elevated after myocardial infarction. Seminoma is associated with increased levels of hCG,
but the increased levels are seen in only about 15% of seminoma cases. Pure seminomas do
not produce AFP, but mixed tumors that contain predominantly seminomatous components
may produce AFP. In NSGCT, approximately 85% to 90% of cases have at least 1 of the 3 tumor
markers elevated. Yolk sac tumors produce AFP, a normal product of the fetal liver and yolk
sac, in about 90% of cases. Choriocarcinoma, a malignant tumor resembling the placental cells,
produces hCG in close to 100% of cases. As is generally true for circulating tumor markers, they
are most useful for monitoring recurrence of disease or as a measure of response to therapy.
Neither hCG nor AFP is useful in screening patients for testicular tumors, and they have a
limited but helpful role in establishing the diagnosis. Higher levels of hCG and LD-1 at the
time of diagnosis are associated with more aggressive cancers and, overall, a less favorable out-
come, and are therefore included in staging classifications for testicular cancer cases. In order
to determine whether a testicular tumor is associated with elevated tumor marker levels, it is
recommended that baseline levels of these 3 markers be measured prior to surgery, since levels
may fall quickly after surgery.
hCG, LD, and AFP are significantly affected by diseases in other organs. LD is found in
all cells. Therefore, damage to any cell can cause increased LD. Since red blood cells contain
LD, a sample collected for LD measurement in which there is red blood cell hemolysis will
show an elevated LD, with much of the LD originating from red blood cells. This is also par-
ticularly problematic in the setting of chemotherapy, where transient LD increases occur from
the cell damage expected from chemotherapy treatment. AFP is also produced by hepatocytes,
as discussed in Chapter 16. Injury to the liver, as occurs with acute or chronic hepatitis, also
commonly causes mild-to-moderate increases in AFP that can lead to suspicion of recurrent
testicular carcinoma.
MALE GONADAL DYSFUNCTION

Description
While complete gonadal failure in men is rare (and is discussed more fully in Chapter 22),
partial androgen deficiency is common with advancing age in men. This has also sometimes
been referred to as “andropause,” and considered by some to be analogous to menopause, the
age-related gonadal failure in women. There are a number of differences between age-related
declines in gonadal function in men and women, however. While gonadal failure is inevitable
7% of men in their 40s in women, not all men develop low levels of testosterone. Menopause typically occurs between
have low androgen levels. the mid-40s and mid-50s, whereas relative androgen deficiency develops over a much broader
However, the figure rises age range in men. Estrogen and progesterone levels fall to extremely low levels in women and
to 30% of men in their are accompanied by high gonadotropin (FSH and LH) levels. However, partial androgen defi-
50s, almost half of those ciency in men is associated with mildly decreased testosterone levels and is usually not associ-
in their 60s, and to 90% of ated with abnormally high gonadotropin levels. According to the Endocrine Society consensus
men in their 80s. guidelines based on testosterone levels in a reference population of healthy young men, only 7%
of men in their 40s have low androgen levels. However, the figure rises to 30% of men in their
50s, almost half of men in their 60s, and to 90% of men in their 80s. Current guidelines indi-
cate that the reference (“normal”) range of testosterone should be based on the values found
in healthy young men, despite some concerns that classifying such a large proportion of tes-
tosterone values seen in older men as abnormal might encourage inappropriate or unneeded
supplemental androgen therapy.
As in women, routine use of hormone replacement in men is controversial. Androgens
increase muscle and bone mass, and may protect against falls and bone fractures. Androgen defi-
ciency can cause mood changes and sexual dysfunction, both of which may respond to androgen
treatment. On the negative side, however, androgens are involved in the pathogenesis of both BPH
and prostate cancer, may lead to a fall in sperm count, and cause undesirable changes in blood
lipids, which may increase the risk of myocardial infarction and stroke. Limited data on safety
and effectiveness of androgen replacement are available, and some level of efficacy that had been
anticipated has not been shown in controlled trials. For example, administration of testosterone
together with sildenafil was not better than sildenafil alone in improving erectile dysfunction in
1 study. Whereas treatment of hypogonadism in young men with primary gonadal failure is ben-
eficial, clear clinical benefits of testosterone therapy in men aged 65 and above with age-related
decreases in testosterone levels have not been demonstrated in randomized trials. Long-term
effects of testosterone therapy on the risk of prostate-related and cardiovascular-related adverse
events remain unknown.
Diagnosis
Laboratory testing for partial androgen deficiency generally begins with measurement of serum
testosterone. Normal levels are often stated to be greater than 250 to 350 ng/dL. Androgen
deficiency is unlikely to be present if the total testosterone is >400 ng/dL. A problem with evalu-
ation of total testosterone levels is that changes in the level of testosterone-binding proteins are
common. Free testosterone, not bound to proteins, contributes significantly more to the biologi-
cal effects of testosterone than does protein-bound testosterone. However, it is widely believed
that testosterone bound to albumin (about 40% of total) may contribute partially to the biological
effects of testosterone. The major testosterone-binding protein, sex hormone-binding globulin
(SHBG), is increased by androgen deficiency, but decreased by obesity, both common problems
in older men. Ideally, free testosterone is quantitated, but some assays for measurement of free
testosterone are unreliable. This has led to the use of tests to determine “bioavailable testosterone,”
which are most helpful in those with testosterone levels between 200 and 400 ng/dL.
Transient decreases in testosterone and gonadotropin levels are commonly seen in persons
who are acutely ill. Testing of gonadal function should be avoided in hospitalized individuals for
that reason. Certain medications, as well as opiates and ethanol, can cause transient decreases
as well. Testosterone secretion has a circadian rhythm, with higher levels in the morning, and
lower concentrations in the afternoon and evening. The reference ranges for expected values of
testosterone are generally based on morning samples. If treatment decisions are to be based on
androgen concentrations, testosterone blood tests should be drawn in the morning, and fasting is
not required. Since testosterone serum levels within an individual are variable, low concentrations
should be confirmed by repeat testing before initiating therapy.
In young men with low testosterone, guidelines suggest measurement of LH. Generally, FSH
follows LH and does not add additional information. It is expected that LH levels are usually
within the reference range in age-related partial androgen deficiency. Very low levels of LH sug-
gest a pituitary or hypothalamic problem, and require further evaluation of pituitary function,
while high levels of LH suggest other causes of a low androgen level.
Infertility (defined as a
failure to conceive after
MALE INFERTILITY 1 year of unprotected
Description intercourse) affects
about 15% of couples
Infertility (defined as a failure to conceive after 1 year of unprotected intercourse) affects about attempting a pregnancy,
15% of couples attempting a pregnancy, and most experts agree that male infertility (alone or in and most experts agree
combination with female infertility) accounts for about half of these cases. The causes of male that male infertility
infertility range from congenital absence of the vas deferens or incomplete spermatogenesis to (alone or in combination
subtle pathologies of sperm shape and function. In contrast, female infertility is usually caused with female infertility)
by tubal blockage, uterine or endometrial abnormalities, or abnormal levels of reproductive accounts for about half of
hormones. Female infertility is relatively easily diagnosed by hormone assays, menstrual cycle these cases.
analysis, and radiologic studies. The male partner in an infertile couple is often overlooked or
incompletely evaluated, even though male factors may be a major contributing factor. Endocrine
abnormalities (eg, isolated androgen deficiency) are exceedingly rare (about 1%) among males
of infertile couples. Since subtle sperm dysfunctions not obvious by microscopic semen analysis
may preclude fertilization, a normal semen analysis (SA) is not necessarily predictive of fertility.
In fact, a complete lack of sperm in semen (azoospermia) is the only highly predictive finding for
infertility by SA.
Absence of spermatozoa in the semen may be caused by either failure of the testes to produce
sperm (ie, male sterility) or a blockage of the excurrent ducts of the male genital tract. The former
is nonobstructive azoospermia (NOA), and the latter, obstructive azoospermia (OA). When azo-
ospermia is discovered by SA, a medical history and physical examination by a qualified physi-
cian is recommended. Testicular biopsy may also be performed to differentiate NOA from OA.
OA may be congenital or acquired. Vasectomy is usually an elective minor surgery leading to
azoospermia for contraception, but the vas or epididymis also may become unknowingly blocked
after a sexually transmitted infection. Azoospermia may persist after vasectomy reversal. Con-
genital bilateral absence of the vas deferens (CBAVD) is found in men carrying one of the many
cystic fibrosis alleles, as well as in men with clinically apparent cystic fibrosis. In all these cases of
OA, spermatozoa are almost always able to be retrieved from the testis by a urologic surgeon. Tes-
ticular sperm are not capable of fertilization except by intracytoplasmic sperm injection, in which
individual sperm are microinjected into an oocyte in an in vitro fertilization (IVF) laboratory. In
the case of a cystic fibrosis carrier, the partner should be checked to determine if she is also a car-
rier for cystic fibrosis so that the couple understands their risk to conceive a child.
Absence of the vasa deferentia may be determined by palpation on physical exam, and may
be suggested by absence of fructose in the semen. Fructose is made exclusively in the seminal
vesicles, and these glands contribute about 60% of the semen volume. Since they are embryonic
outgrowths of the vasa deferentia, they are usually absent in men with CBAVD.
Two reasons for NOA are absence of germ cells in the testis (Sertoli-only syndrome) and
failure of spermatogenesis (only mitotic cells or no late stages of sperm production). These condi-
tions may be genetic, due to failure of primordial germ cells to migrate to the testis, or acquired
through exposure to toxicants. In addition, microdeletions in the Y chromosome in specific
regions are associated with NOA or severe oligozoospermia. Testosterone or androgen therapy
(see above) for low testosterone or bodybuilding will often result in azoospermia or oligozoosper-
mia due to inhibition of the hypothalamic–pituitary axis, decreased LH, and subsequent lowering
of the high intratesticular:plasma ratio of testosterone required for sperm production. Men with
testicular and other cancers may exhibit NOA or oligozoospermia. Conversely, men with NOA or
oligozoospermia are at increased risk for later development of testicular cancer.
Spermatozoa may not be entirely missing in NOA. A few stem cells throughout the testis
may be present, although a random biopsy may not detect any. In such cases, exceedingly low
numbers of sperm may be present in the semen, possibly on a cyclical basis. Cryptozoospermia is
the presence of very rare sperm in the semen. Often, high levels of FSH are associated with NOA,
due to dysfunctional Sertoli cells and lack of the Sertoli hormone inhibin. However, high FSH has
not proven to be a good indicator of the absolute absence of spermatogenesis. The surgical tech-
nique known as microsurgical testicular sperm extraction has proven to be useful in these cases.
Under the surgical microscope, a few seminiferous tubules might be located that have complete
spermatogenesis. Highly trained staff in the andrology or embryology laboratory are needed to
identify and recover sperm from the testis in these cases.
In other cases of male infertility, sperm will be present in the semen. The human, unlike
almost any other species, produces spermatozoa with numerous defects. “Normal” values
for human SA are both surprisingly poorly established and highly variable from 1 ejaculate to
another, and between different men. Although a typical ejaculate contains 200 to 300 million
sperm, fertilization only takes 1, and at the time of fertilization there are only about a dozen
sperm associated with the egg. If only 1% of sperm are functionally and structurally normal, then
the typical ejaculate will contain about 2 to 3 million of these “good sperm.” Apparently this is
enough to help propagate the human population. SA helps identify men who have a few “good
sperm,” and diagnose specific sperm problems that may permit therapy or advanced reproductive
techniques, or help guide decisions by the couple.
Diagnosis and Laboratory Test Interpretation Semen analysis (SA) is

SA is the cornerstone of male infertility diagnosis, and should be performed early in the evalu- the cornerstone of male
ation of the infertile couple. Collection of a semen sample should take place after 2 to 5 days infertility diagnosis, and
should be performed
of sexual abstinence. Longer abstinence is associated with lower motility and higher leukocyte
early in the evaluation
counts, and shorter periods usually lead to lower volumes and sperm counts. A sterile plastic
of the infertile couple. If
container, known not to be toxic to sperm, is required. Usually a sterile urine collection cup is
SA is abnormal, it should
satisfactory. If the sample is not collected in a special collection room near the lab, it should be be repeated after at least
transported in a tightly sealed collection cup in a sealed plastic bag, protected from light, heat, 1 month. SA parameters
and cold. A temperature between room temperature and body temperature is adequate. Seminal can vary substantially
fluid characteristics are measured after allowing semen to liquefy (due to the enzymatic action of within an individual.
PSA) for 15 to 30 minutes at 37°C or at room temperature. Analysis should be completed within Therefore, a single
60 minutes after ejaculation. In vivo, sperm typically swim out of semen into the cervical mucus abnormal specimen
within a half hour. Semen may contain hepatitis, HIV, and other pathogens. Basic precautions for should not be used to
handling potentially infectious body fluids must be followed. establish a diagnosis.
The main tests in SA are ejaculate volume, sperm count, sperm motility, sperm morphol-
ogy, and leukocyte count. Aside from azoospermia, findings that may contribute to a couple’s
infertility include low sperm concentration or total number of sperm (oligozoospermia), poor
sperm motility (asthenozoospermia), very poor sperm morphology (teratozoospermia), or any
combination of the 3. Additionally, a high number of neutrophils or macrophages in the semen
(leukocytospermia) may affect sperm function by inducing oxidative stress through the genera-
tion of reactive oxygen species. Leukocytes are likely present in all semen samples. There is lack of
consensus on the number that is considered abnormal, with authorities suggesting various cutoffs
from 200,000 to 1 million per mL of semen.
A semen fructose analysis may be considered if there is unexpected azoospermia. Sperm
viability is important to determine when motility is very low. Sperm agglutination and the pres-
ence of antisperm antibodies may indicate an immunologic basis for infertility. These highly spe-
cialized tests typically are performed in laboratories certified specifically for SA.
Interpretation of sperm morphology is controversial, largely because of a lack of support-
ing data relating sperm morphology to fertility. Very few human sperm are “perfect”; in fact, if
at least 4% of sperm have normal morphology, then the specimen morphology is generally con-
sidered to be normal. The clinical significance of sperm morphology is best understood when
most of the sperm have specific defects. The absence of acrosomes (the specialized structure at
the tip of the sperm head) is associated with inability to fertilize eggs, and, sometimes, inability
to activate oocytes to complete meiosis. Abnormal midpieces or tails may be associated with
poor motility.
If SA is abnormal, it should be repeated after at least 1 month. SA parameters can vary sub-
stantially within an individual. Therefore, a single abnormal specimen should not be used to
establish a diagnosis.
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C H A P T E R
Female Genital System

Stacy E.F. Melanson and Ann M. Gronowski 20
LEARNING OBJECTIVES
1. Understand the clinical utility of human chorionic gonadotropin (hCG) results
in pregnancy, normal and ectopic, spontaneous abortion (miscarriage), and
gestational trophoblastic disease.
2. Learn how to diagnose common complications of pregnancy, notably
preeclampsia, eclampsia, HELLP syndrome, and fatty liver associated
with pregnancy.
3. Understand the causes and the diagnosis of female infertility.
CHAPTER OUTLINE
Introduction 405 Gestational Trophoblastic Disease 410
Normal Pregnancy 405 Preeclampsia and Eclampsia 410
Maternal Serum Screening 407 HELLP Syndrome 411
Ectopic Pregnancy 408 Fatty Liver of Pregnancy 411
Spontaneous Abortion (Miscarriage) Female Infertility 411
and Recurrent Abortion 409
INTRODUCTION
Clinical laboratory testing is useful for the diagnosis and management of pregnancy and
infertility, and such testing is reviewed in this chapter. Gestational diabetes mellitus (GDM) is
discussed in Chapter 17, and hemolytic disease of the newborn (HDN) is found in Chapters 7
and 12. Female physiology and biochemistry including amenorrhea are discussed in Chapter 22.
The female genital tract is also a common site for infections, which may be sexually transmitted,
and it is a common site for tumors. The infections are presented in Chapter 5, and tumor
descriptions are found in textbooks of anatomic pathology.
NORMAL PREGNANCY
Description
Normal pregnancy lasts approximately 40 weeks, as dated from the first day of the previous men-
strual period, and is typically divided into 3 intervals or trimesters each lasting approximately
13 weeks. Approximately 5 days after fertilization, a blastocyst implants in the uterus. Trophoblast
cells of the blastocyst invade the endometrium with chorionic villi leading to a placenta and the
forming embryo surrounded by amniotic fluid. The placenta nourishes the embryo and produces
hormones vital to pregnancy such as human chorionic gonadotropin (hCG), progesterone, estra-
diol, estriol, and estrone. The amniotic fluid protects the embryo and changes composition as
the pregnancy progresses. The embryo undergoes rapid cell division, differentiation, and growth
in the first trimester (0-13 weeks). By 10 weeks, most major structures are formed resulting in a
405
406 CHAPTER 20 Female Genital System
TABLE 20–1 Routine Testing in Normal Pregnancy

Test Comments
Chemistry
hCG Should double every 1.5-2 days for the first 8 weeks
First-trimester screen (free beta To assess for trisomy 21

hCG, PAPP-A)
Second-trimester “quad” screen To assess for trisomy 21, neural tube defects, and other fetal anomalies
(hCG, AFP, estriol, inhibin A)
Glucose To assess for gestational diabetes mellitus
Blood bank
Type and screen To assess for the risk of hemolytic disease of the newborn; includes blood
type (ie, A, B, AB, O), Rh typing (ie, negative or positive), and antibody screen
Microbiology
RPR or treponemal antibody To screen for syphilis
Hepatitis B surface antigen To assess for active hepatitis B
HIV antibody To assess for exposure to HIV
Group B Streptococcus (GBS) To assess for GBS in the third trimester and if present prevent
cervical culture transmission to fetus during delivery
hCG, human chorionic gonadotropin; PAPP-A, pregnancy-associated plasma protein-A; AFP, alpha-fetoprotein.
Pregnancy has an effect fetus. The second trimester (13-26 weeks) is associated with rapid fetal growth. Completion of
on many laboratory tests, maturation occurs in the third trimester (26-40 weeks) resulting in a term pregnancy between
and these alterations 37 and 42 weeks.
should be considered
when interpreting
Diagnosis
laboratory tests from
pregnant women. Once pregnancy has been achieved, several laboratory tests are routinely performed to help ensure
an optimal maternal and fetal outcome (Table 20–1). Most testing in pregnancy is performed on
maternal serum because it is easy to obtain and provides minimal risk to the pregnancy, but
maternal urine and amniotic fluid specimens may also be necessary. Of note, pregnancy has an
effect on many laboratory tests, other than those used to diagnose and manage pregnancy, and
these alterations should be considered when interpreting laboratory tests from pregnant women
(Table 20–2).
TABLE 20–2 Effects of Pregnancy on Select Laboratory Tests

Test Result in Pregnancy Comments
Hematocrit Decreased Due to an increased plasma volume
Coagulation factors Several factors increase; some do The overall effect is an increased
not change; Factor XI decreases thrombotic risk
Lipids (triglycerides, cholesterol) Increased
Thyroxine-binding globulin, Increased Patient remains euthyroid

total T3 and T4
Alkaline phosphatase activity Increased Due to production of placental,

heat-stable, alkaline phosphatase
BUN, creatinine Slightly decreased Due to increased glomerular

filtration rate
1,25-Dihydroxyvitamin D Increased Due to increased calcium and

transfer of calcium to fetus
Parathyroid hormone Increased Ionized calcium remains normal

CHAPTER 20 Female Genital System 407
hCG is one of the most commonly ordered tests in pregnancy. It is heterodimer composed of
2 nonidentical nonconvalently bound glycoprotein subunits, alpha and beta, that is synthesized
by the trophoblasts of the placenta. Only the intact molecule is biologically active. A single gene
for the alpha subunit of all 4 glycoprotein hormones (thyroid-stimulating hormone [TSH], lutein-
izing hormone [LH], follicle-stimulating hormone [FSH], and hCG) is found on chromosome
6. hCG stimulates the LH receptor on the corpus luteum to produce progesterone that helps to
prevent pregnancy loss.
Detectable amounts of hCG (>2-5 IU/L, depending on the assay) are present in serum 8 to Detectable amounts
11 days after conception. Qualitative urine hCG tests are usually sufficient for screening, but the of hCG are present in
detection limits of qualitative tests range from 20 to 50 IU/L, limiting their use to the time follow- serum 8 to 11 days after
ing a missed menstrual period or greater than 14 days after conception. As opposed to qualitative conception.
testing, quantitative testing offers sensitivity as low as 2 to 5 IU/L and serial measurements may
be helpful to reveal problems in a pregnancy. In normal pregnancies, hCG doubles every 1.5 to
2 days for the first 8 weeks (Table 20–1) and peaks around 100,000 to 500,000 IU/L.
Blood and urine contain many hCG variants, including free subunits, and hyperglycosylated
and nicked forms. After 5 weeks of gestation the predominant hCG form in urine is the hCG beta
core fragment. In addition, the hCG glycosylation changes as the pregnancy progresses. Quan-
titative hCG immunoassays typically measure total hCG beta concentrations using 2 antibod-
ies against different regions of the beta subunit. False-negative hCG results can be seen in early
pregnancy or when hCG concentrations are very high, causing a hook effect. False-positive results
can be caused by heterophilic antibodies and by advanced maternal age (from pituitary hCG).
Other additional routine testing in pregnancy is performed to screen for common and/or
treatable pregnancy complications such as gestational diabetes (see Chapter 17), hemolytic disease
of the newborn (see Chapters 7 and 12), and infection (see Chapter 5) (Table 20–1).
MATERNAL SERUM SCREENING

Description
Maternal serum screening can be performed to identify individuals who need further diagnostic
evaluation for fetal anomalies such as neural tube defects (NTDs), trisomy 21/Down syndrome,
and trisomy 18. NTDs result from failure of fusion of the neural plate and failure of complete cov-
ering by the 27th day post conception. The extent and location of neural tissue exposed indicates
the severity of the defect (ie, anencephaly, meningomyelocele, and encephalocele). The result of
a NTD is a direct communication of the amniotic fluid with fetal plasma proteins, and release of Maternal serum screening
alpha-fetoprotein (AFP) into amniotic fluid and maternal serum. Rates of NTDs have decreased can be performed to
due to the addition of folic acid to grain, as well as initiation of recommendations for folic acid identify individuals
supplementation prior to conception. Trisomy 21 or Down syndrome is caused by an extra copy who need further
of chromosome 21 and is the most frequent chromosomal disorder among live-born children diagnostic evaluation
(1/600 to 1/800 live births). Risk factors for Down syndrome include advanced maternal age, the for fetal anomalies such
birth of a previously affected child, and balanced parental structural rearrangement of chromo- as neural tube defects
some 21. Affected children suffer from mental retardation, hypotonia, congenital heart defects, (NTDs), trisomy 21/
and a flat facial profile. The main phenotypic features of trisomy 18 include hypertonia, promi- Down syndrome, and
nent occiput, small mouth, micrognathia, short sternum, and horseshoe kidney. trisomy 18.
Screening
An additional discussion of maternal screening for Down syndrome is found in Chapter 7.
Screening for NTDs is done at 15 to 22 weeks gestation by measurement of serum AFP that
is expressed as a multiple of the median population (MoM). MoMs greater than 2 or 2.5 are con-
sidered abnormal and should be followed up by high-resolution ultrasound or measurement of
amniotic fluid AFP and acetylcholinesterase.
Sequential serum screening is performed to screen for trisomies and combines first- and
second-trimester testing. First-trimester testing is performed between 10 and 14 weeks and
includes measurement of hCG and pregnancy-associated plasma protein-A (PAPP-A) as well as
an ultrasound measurement for infant nuchal translucency (NT). Specific training is required of
operators for determination of NT. This procedure is highly operator dependent. Free beta hCG
testing is more accurate than intact hCG testing in the first trimester and is used instead of intact
hCG for the first-trimester screen. PAPA-A is a protein produced by the placenta. Elevated hCG,
decreased PAPP-A, and increased NT are seen in pregnancies affected by trisomy 21.
In the sequential screen, first-trimester screening is followed up with second-trimester
screening at 15 to 22 weeks, with measurement of serum AFP, hCG, estriol, and inhibin A (ie,
the quad screen). AFP is the most abundant serum protein in the fetal circulation. Maternal
serum AFP is detectable at 10 weeks and peaks at 15 to 20 weeks. Estriol is the predominant
estrogen of pregnancy and also the most difficult to measure because of low concentration and
limited stability. Inhibin A, secreted by the ovaries and placenta, is a glycoprotein that inhibits
FSH. Concentrations of individual analytes from the first- and second-trimester screens and
NT measurements are combined into a risk assessment algorithm that adjusts for gestational
age, maternal weight, number of fetuses, and presence or absence of diabetes mellitus. The most
frequent causes of abnormal results include incorrect dating, the presence of twins, and fetal
demise. For this reason, an ultrasound confirming gestational age is the first line of testing in a
patient with apparently increased risk. Fetal karyotyping is necessary to confirm chromosomal
abnormalities.
Second trimester-only testing can be done and should utilize the quad screen. Elevated
hCG, increased inhibin A, decreased estriol, and decreased AFP are seen in pregnancies affected
by trisomy 21, while all 4 analytes in the quad screen are decreased in pregnancies affected by
trisomy 18. Patients at risk should undergo further diagnostic evaluation as described with
sequential screen.
Recently, new methods have been developed for trisomy 21 screening using circulating fetal
DNA. These methods utilize “massively parallel genomic sequencing” (MPGS). DNA fragments
are isolated from a sample of maternal blood, which contains a mixture of maternal DNA and
infant DNA. The DNA fragments are amplified and then sequenced. The number of sequences
that originate from a particular chromosome is counted and tabulated for each chromosome. If a
fetus has an extra chromosome, then the percentage of DNA fragments from that chromosome is
higher than expected. These tests have been shown to have excellent detection rates (∼99%) with
very low false-positive rates (∼0.2%). Currently, MPGS is only validated for the detection of Down
syndrome and not other fetal aneuploidies such as trisomy 18 or trisomy 13. The test does have
the capability of detecting these disorders and, if they are detected, the results are reported. How-
ever, because the test has not been thoroughly investigated for detecting trisomy 18 or trisomy
13, a negative result does not rule out their presence. Also, the test does not detect open NTDs
(eg, spina bifida), for which screening usually involves biochemical (not genetic) screening tests.
The American College of Obstetricians and Gynecologists (ACOG) cautions that these tests
are screening tests, not diagnostic tests. They also recommend that DNA-based screening tests be
performed only on women who are at increased risk of having a fetus with aneuploidy, including
Three classic symptoms women with: maternal age 35 years or older at delivery, fetal ultrasound findings suggesting aneu-
of an ectopic pregnancy ploidy, a previous aneuploid pregnancy, or abnormal biochemical screening test results.
include lower abdominal
pain, vaginal bleeding,
and an adnexal mass. ECTOPIC PREGNANCY
However, only 25% of
women with ectopic Description
pregnancy have these Ectopic pregnancies arise if the fertilized egg implants in a location other than the body of the
symptoms at the time uterus, primarily in the fallopian tube. Of all reported pregnancies, 1.3% to 2% are extrauterine.
of presentation, making The nonuterine location of implantation prevents normal development. Despite increased aware-
laboratory testing and ness and improved diagnostic modalities, such as serial hCG and transvaginal ultrasound, ectopic
transvaginal ultrasound pregnancies are the leading cause of maternal death in the first trimester. Risk factors for ectopic
examination essential pregnancy include tubal damage from either infection or disease, smoking, infertility, and previ-
for diagnosis and ous miscarriage.
management.
Diagnosis
Three classic symptoms of an ectopic pregnancy include lower abdominal pain, vaginal bleeding,
and an adnexal mass. However, only 25% of women with ectopic pregnancy have these symptoms
at the time of presentation, making laboratory testing and transvaginal ultrasound examination
TABLE 20–3 Abnormal Pregnancy Conditions

Condition Laboratory Diagnosis
Ectopic pregnancy Slow rate of increase in hCG
Gestational trophoblastic disease hCG concentration greater than that expected for gestational age;
rate of increase may be accelerated as well
Preeclampsia Modest increase in AST and ALT (4-10× upper limit)

>0.3 g/L protein in 24-h urine
>1.0 g/L protein in random specimen
HELLP syndrome Decreased platelets (<100,000/μL)

Elevated LDH (>600 IU/L)
Elevated ALT and AST (200-700 IU/L)
Fatty liver of pregnancy Mild increase in AST and ALT (AST > ALT)
Serum bilirubin >6 mg/dL
Hypoglycemia
Increased uric acid
Prolonged PT and PTT
Low fibrinogen
hCG, human chorionic gonadotropin; PT, prothrombin time; PTT, partial thromboplastin time.
essential for diagnosis and management. In ectopic pregnancies, abnormal concentrations of hCG
are present (Table 20–3). hCG concentrations in ectopic pregnancy range from undetectable to
200,000 IU/L, depending on the size and viability of the trophoblastic tissue. Therefore, the abso-
lute concentration of hCG is not very useful in the diagnosis of ectopic pregnancy. Many utilize
a so-called discriminatory zone of hCG in which a fetus should be visible when hCG concentra-
tions are >2000 IU/L. However, recent studies suggest that the discriminatory zone is not very
reliable and can lead to misdiagnosis. Instead serial testing reveals rates of hCG increase as slow
as 35% over 2 days in ectopic pregnancy. Medical therapy with methotrexate or surgery is required
to prevent rupture and significant hemorrhage.
SPONTANEOUS ABORTION MISCARRIAGE

AND RECURRENT ABORTION
Description
Spontaneous abortion or miscarriage refers to a pregnancy that ends spontaneously before the
fetus has reached a viable gestational age. The most common complication of early pregnancy is
spontaneous abortion, and it occurs in approximately 10% to 20% of all recognized pregnancies
under 20 weeks gestation. The percentage increases if unrecognized or subclinical pregnancies
are included. Risk factors include advanced maternal age, previous miscarriage, smoking, and
alcohol or drug consumption. Chromosomal abnormalities account for approximately 50% of all
miscarriages.
Recurrent spontaneous abortion is defined as 3 or more consecutive intrauterine pregnancy The most common
losses prior to 20 to 24 weeks of gestation. It affects up to 1% to 5% of fertile couples. Primary complication of early
aborters have had no live births, while secondary aborters were able to achieve at least 1 successful pregnancy is spontaneous
pregnancy. Assisted reproduction technologies are much less effective in women with recurrent abortion, and it occurs
fetal losses compared with those with infertility. in approximately 10%
to 20% of all recognized
pregnancies under
Diagnosis
20 weeks gestation.
Women experiencing a miscarriage may present with a history of amenorrhea, vaginal bleed-
ing, and lower abdominal pain. Serial measurements of hCG concentration in conjunction with
physical examination and ultrasonography can be helpful in the diagnosis of spontaneous abor-
tion. Decreasing hCG concentrations are consistent with a spontaneous abortion or nonviable
pregnancy. Patients with a confirmed miscarriage can be managed expectantly, medically with
misoprostol, or surgically. Following treatment, hCG concentrations can be monitored until the
TABLE 20–4 Laboratory Evaluation of Recurrent Spontaneous Abortion

Analysis Purpose
Parental and fetal tissue karyotype Tests for chromosomal abnormalities
LH, FSH, thyroid-stimulating hormone, prolactin, cortisol Tests for endocrine abnormalities
Thrombotic risk factors including protein C, protein S, and Tests for thrombophilic disorders
antithrombin deficiencies, Factor V Leiden and prothrombin gene
20210 mutations, lupus anticoagulant, anticardiolipin antibodies
Antithyroid antibodies Tests for autoimmune factors
Endometrial biopsy Tests for luteal phase defect or

inadequate endometrial maturation
Glucose, hemoglobin A1c Tests for diabetes
concentration is undetectable to confirm complete expulsion of products of conception. It may

take 30 to 60 days before serum hCG concentrations are undetectable. The etiology of recurrent
loss is often unclear, but can include genetic, anatomic, hormonal, thrombotic, placental, infec-
tious, environmental, or psychological causes. Immunological factors may also play a role. Fol-
lowing a detailed history, physical examination, and radiological studies, additional laboratory
tests may be helpful in determining the cause of the recurrent loss (Table 20–4). Of all the etio-
logic factors, only parental genetics has been shown to be a definitive cause of recurrent loss.
Although uterine abnormalities, antiphospholipid antibodies, the Factor V Leiden mutation, and
other thrombotic risk factors (see Chapter 11) are definitely associated with recurrent loss, there
is not sufficient proof of a causative role.
GESTATIONAL TROPHOBLASTIC DISEASE

Description
Gestational trophoblastic diseases are a spectrum of disease processes originating from the pla-
centa that include hydatidiform mole, invasive mole, and choriocarcinoma. Malignant gestational
trophoblastic diseases have the potential for local invasion and metastasis.
Hydatidiform moles are the most common and occur in 1 of 600 therapeutic abortions and
1 of 1100 pregnancies. Approximately 20% of patients will be treated for malignant sequelae after
evacuation of a hydatidiform mole. Gestational choriocarcinoma occurs in approximately 1 in
30,000 pregnancies.
Diagnosis
Gestational trophoblastic diseases are usually diagnosed early in pregnancy. Patients pres-
ent with abnormal bleeding and vague complaints. Ultrasound and serum hCG testing are
used to make the diagnosis of gestational trophoblastic diseases. Ultrasound findings include
the absence of a fetal heartbeat. hCG testing reveals greatly elevated hCG concentrations
(Table 20–3). Dilation and evacuation (D&E) procedures are performed to treat patients. Serial
hCG measurements should be performed after treatment to ensure complete removal of tumor
Preeclampsia is a and monitoring of disease for recurrence. Chemotherapy may be necessary in cases of malig-
multisystem disorder nant transformation.
of unknown etiology,
and it is a major cause
of morbidity and PREECLAMPSIA AND ECLAMPSIA
mortality in pregnancy.
Preeclampsia is diagnosed
Description
by the occurrence of Preeclampsia is a multisystem disorder of unknown etiology, and it is a major cause of morbidity
new hypertension and and mortality in pregnancy. Patients develop elevated blood pressure and proteinuria. In
proteinuria in the second addition, coagulopathies, impaired liver function, renal failure, and cerebral ischemia may occur.
half of pregnancy. Preeclampsia occurs in 2% to 8% of pregnancies. Eclampsia, in which women with preeclampsia
have accompanying seizures, occurs less frequently. Eclampsia is more serious and carries a
higher morbidity and mortality rate. Treatment includes controlling symptoms until delivery.
Diagnosis
Preeclampsia is diagnosed by the occurrence of new hypertension and proteinuria in the sec-
ond half of pregnancy. Hypertension in pregnancy is defined as a persistent systolic blood pres-
sure ≥140 mm Hg and/or a diastolic blood pressure ≥90 mm Hg. Proteinuria in preeclampsia
is >300 mg/L protein in a 24-hour urine specimen or >1 g/L protein in a single urine specimen
(Table 20–3). It should be demonstrated that seizures associated with eclampsia are not explained
by a neurological disorder such as epilepsy.
HELLP SYNDROME
Description
The HELLP syndrome involves hemolysis, elevated liver enzymes, and a low platelet count. The
syndrome can occur during pregnancy, typically between weeks 27 and 36, or in association with
preeclampsia; it can also occur postpartum.
Diagnosis
The HELLP syndrome and preeclampsia have similar clinical presentations. A low platelet
count and abnormal liver enzymes are important to make a diagnosis of the HELLP syndrome
(Table 20–3). The hemolysis in the HELLP syndrome is microangiopathic, and this results in
schistocytes on the peripheral blood smear, an elevated indirect bilirubin, and an increased lac-
tate dehydrogenase (LD) activity.
FATTY LIVER OF PREGNANCY

Description
Approximately 1 in 13,000 pregnancies is affected by fatty liver of pregnancy. First pregnancies
and multiple gestation pregnancies are at a higher risk. Symptoms, which are nonspecific and
include nausea and vomiting, right upper quadrant pain, and lethargy, typically begin around the
36th week of gestation. Liver biopsies show accumulation of microvesicular fat, which may be
caused by a defect in mitochondrial beta oxidation of fatty acids or a long-chain 3-hydroxyacyl
CoA dehydrogenase deficiency. Treatment involves immediate delivery to prevent fulminant
hepatic failure requiring liver transplantation. Recurrence in subsequent pregnancies is rare.
Diagnosis
The diagnosis is made using both clinical symptoms and laboratory results. Although liver biopsy
is virtually diagnostic in the setting of pregnancy, it is rarely necessary. The laboratory test abnor-
malities include mild elevations in liver enzymes (AST > ALT), increased bilirubin, hypogly-
cemia, and hyperuricemia (Table 20–3). Abnormal coagulation test results, as indicated by a
prolonged prothrombin time, a prolonged partial thromboplastin time, and a low fibrinogen, are
found in acute fatty liver of pregnancy, but not in HELLP syndrome, and this helps differentiate
the 2 conditions.
FEMALE INFERTILITY
Infertility is defined as
Description
the inability to achieve
Infertility is defined as the inability to achieve a successful pregnancy following 1 year of unpro- a successful pregnancy
tected intercourse. It is estimated that 15% to 25% of couples will experience an episode of infer- following 1 year of
tility. Couples with primary infertility have had no previous successful pregnancies. Couples with unprotected intercourse.
secondary infertility had prior pregnancies, but are currently unable to conceive. Both primary It is estimated that 25% of
and secondary infertility have common causes, most often problems with the hypothalamic– couples will experience an
pituitary–gonadal axis. episode of infertility.
Midluteal progesterone Diagnosis

concentrations greater Factors contributing to female infertility include ovarian, hormonal, tubal, cervical, uterine,
than 10 ng/mL indicate psychosocial, iatrogenic, and immunological factors. Ovulatory disorders, such as hypergo-
normal ovulation
nadotropic hypogonadism and hypogonadotropic hypogonadism, are the most common and
while concentrations
are described in more detail in Chapter 22. Other disorders such as polycystic ovarian disease,
less than 10 ng/mL
obesity, thyroid dysfunction, androgen excess, and liver dysfunction can also contribute.
suggest anovulation,
inadequate luteal phase Following a detailed history and physical examination, laboratory evaluation of female infertility
progesterone production, is performed. Midluteal progesterone concentrations greater than 10 ng/mL indicate normal
or inappropriate timing of ovulation while concentrations less than 10 ng/mL suggest anovulation, inadequate luteal phase
sample collection. Serum progesterone production, or inappropriate timing of sample collection. Serum concentrations of
concentrations of FSH and FSH and estradiol can be measured on day 3 of the menstrual cycle to indicate ovarian reserve.
estradiol can be measured TSH and prolactin (PRL) can be used to assess for thyroid or pituitary dysfunction.
on day 3 of the menstrual
cycle to indicate ovarian
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C H A P T E R
Breast
Karin E. Finberg 21
LEARNING OBJECTIVES
1. Learn the tissue- and serum-based biomarkers in breast cancer.
2. Understand how the individual biomarkers are used clinically.
CHAPTER OUTLINE
Introduction 413 Hereditary Breast and Ovarian
Breast Cancer 413 Cancer Syndrome 417
Laboratory Testing 414 Other High-penetrance Cancer
Tissue-based Biomarkers Predisposition Genes 418
in Breast Cancer 414 Li-Fraumeni Syndrome 418
Serum-based Biomarkers Cowden Syndrome 418
in Breast Cancer 416 Peutz-Jeghers Syndrome 419
INTRODUCTION
This chapter focuses on laboratory testing relevant to breast cancer. Infections of the breast are
included in Chapter 5.
BREAST CANCER
Description
Cancers of the breast constitute a major cause of mortality in women of Western countries. In the
United States, the lifetime probability that a woman will develop breast cancer is 1 in 8. Breast
cancer accounts for 29% of new cancer cases and 14% of cancer deaths in American women.
About 1% of breast cancers occur in males. The risk of developing breast cancer is influenced by
several factors. These factors include increased age, family history of breast cancer (especially in
a first-degree relative), hormonal factors (early age at menarche, older age of menopause, older
age at first full-term pregnancy, fewer number of pregnancies, and use of hormone replacement
therapy), clinical factors (high breast tissue density and benign breast diseases associated with
atypical hyperplasia), obesity, and alcohol consumption. Since 1990, the mortality rate associated
with female breast cancer has decreased in the United States, a decline that has been attributed to
both therapeutic advances and early detection.
For localized breast cancer, primary treatment typically consists of either breast-conserving
surgery and radiation or mastectomy. Most patients with invasive breast cancer subsequently
receive systemic adjuvant chemotherapy and/or hormone therapy, both of which have been
shown to reduce systemic recurrence and breast cancer-related mortality. However, the fact
that some patients who lack lymph node involvement are cured by the combination of sur-
gery and radiotherapy suggests that adjuvant treatment may not be necessary in all cases.
413
414 CHAPTER 21 Breast
Therefore, to rationally administer adjuvant therapy to patients with local disease, several
prognostic factors are considered to assess the risk for recurrence. These prognostic factors
include tumor size, axillary node involvement, histological type, cytological grade, lymphatic
and vascular invasion, and certain biomarkers associated with breast cancer.
While adjuvant therapy improves patient outcomes, 25% to 30% of women with lymph
node-negative and at least 50% to 60% of women with node-positive disease develop recurrent
or metastatic disease. Metastatic breast cancer is currently regarded as incurable. Therapeutic
options for metastatic disease include chemotherapy, hormone therapy, and molecularly tar-
geted therapies. In the context of metastatic disease, information gained from serial monitoring
of tumor markers detected in the serum may contribute to decisions to continue or terminate a
particular treatment.
LABORATORY TESTING
Tissue-based Biomarkers in Breast Cancer
Assessment of biomarkers in tissue obtained from the patient’s breast tumor is routinely performed
to obtain prognostic information and to guide therapy.
Estrogen Receptor (ER) and Progesterone Receptor (PR)

ER and PR are intracellular receptors that bind to lipid-soluble steroid hormones that diffuse into
target cells. Following ligand binding, 2 receptor subunits dimerize to form a single, functional
Assessment of biomarkers DNA-binding unit that binds to specific DNA target sequences to induce transcription of target
in tissue obtained from genes. There are 2 different forms of the ER, termed ER-α and ER-β, which are encoded by sepa-
the patient’s breast tumor rate genes. Clinical assays assess ER-α, the classical form of the receptor. PR has 2 isoforms that
is routinely performed differ in molecular weight but are encoded by a single gene.
to obtain prognostic Measurement of the ER and PR status of the tumor is recommended in all patients with
information and to breast cancer. ER expression is present in approximately 70% of breast cancers, is associated with
guide therapy. a favorable prognosis, and suggests that the growth of the tumor may be estrogen-dependent. The
primary purpose of determining ER and PR status in breast cancers is to identify those patients,
in both the adjuvant and metastatic settings, who are likely to respond to endocrine treatments.
These treatments act by either preventing the formation of estrogen from its precursors or block-
ing estrogen from binding to its receptors. Endocrine treatments include tamoxifen, ovarian abla-
tion (surgical or chemical), aromatase inhibitors (anastrozole, letrozole, and exemestane), and
irreversible ER inhibitors (eg, fulvestrant). In patients with ER-positive tumors, 5 years of adju-
vant treatment with tamoxifen significantly reduces annual death rates from breast cancer, while
in patients with ER-negative tumors, tamoxifen shows little effect on recurrence or death, and it
does not significantly modify the effects of polychemotherapy.
ER/PR status is routinely assessed by immunohistochemistry (IHC) in the clinical setting.
IHC evaluates the percentage of cells with nuclear ER/PR. The intensity of staining is also
recorded as a measure of assay quality. The use of validated antibodies is required, and a positive
control (ie, a control tissue with tumor cells known to express the respective receptor) must be
examined in parallel. A tumor is scored as positive for either ER or PR if ≥1% of tumor cell
nuclei are immunoreactive. A tumor is scored as negative for ER or PR if <1% of tumor cell
nuclei are immunoreactive in the presence of demonstrable staining in adjacent normal breast
epithelial cells, which serves as an internal positive control. If tumor cells are not found to be
immunoreactive and the specimen lacks an appropriately stained internal control, the tumor is
scored as uninterpretable for ER or PR. For optimal results, breast resection specimens should be
fixed within 1 hour. Fixation should be performed in 10% neutral buffered formalin for at least
6 hours and for not more than 72 hours in order to preserve ER and PR epitope recognition and
thus avoid false-negative results.
HER2
HER2 (also known as ERBB2 and NEU) is a proto-oncogene located at chromosome 17q11 that
is a member of the epidermal growth factor receptor (EGFR) family. Like other EGFR family
CHAPTER 21 Breast 415
members, HER2 is a transmembrane receptor with cytoplasmic tyrosine kinase activity. Dimer-
ization of the receptor leads to phosphorylation of a variety of substrates, resulting in the activa-
tion of intracellular signaling pathways important for cell proliferation and survival.
While normal cells contain 2 copies of the HER2 gene (1 copy on each chromosome 17),
in approximately 10% to 25% of breast cancers HER2 gene copy number is increased at least
2-fold relative to the number of copies of chromosome 17, a phenomenon termed gene amplifica-
tion. Gene amplification results in overexpression of the HER2 protein at the cell surface, which
in turn promotes tumor cell proliferation and survival. Tumors that overexpress HER2 behave
more aggressively than those lacking overexpression, and they are associated with poorer clinical
outcomes.
Assessment of HER2 status of the tumor is recommended in all patients with invasive breast
cancer. The primary purpose of HER2 testing is to identify those patients with early or advanced
breast cancer who are eligible for treatment with trastuzumab, a recombinant monoclonal anti-
body that recognizes HER2. Although its exact mechanism of action remains to be fully elucidated,
trastuzumab has been shown in both in vitro assays and animal studies to inhibit proliferation
of human tumor cells that overexpress HER2. In patients with HER2-positive early stage breast
cancer, the addition of trastuzumab to adjuvant chemotherapy significantly improves disease-free
and overall survival. Additionally, in patients with HER2-positive metastatic breast cancer, the
addition of trastuzumab to adjuvant chemotherapy significantly increases the time until disease
progression. Because a small percentage of patients treated with trastuzumab develop cardiotox- The primary purpose
icity, the elimination of false-positive HER2 results is important so that patients are not exposed of HER2 testing is to
to this risk unnecessarily. identify those patients
HER2 status is routinely assessed in formalin-fixed tissues by either fluorescence in situ with early or advanced
hybridization (FISH) or IHC. FISH assesses HER2 status at the DNA level. A fluorescent- breast cancer who are
labeled nucleic acid probe that recognizes the HER2 gene on chromosome 17 is hybridized on eligible for treatment
with trastuzumab, a
tissue sections, and the average number of HER2 signals per nucleus is determined in areas
recombinant monoclonal
of invasive tumor. In some assay systems, an additional probe that recognizes the centro-
antibody that recognizes
meric region of chromosome 17 (CEP17) (and which is labeled with a different fluorophore) is HER2.
included to allow the ratio of the average number of copies of HER2:CEP17 (the “FISH ratio”)
to be calculated. Tumors with intermediate results are considered equivocal for gene amplifi-
cation; in these cases, IHC for HER2 protein may be performed to resolve HER2 status. Chro-
mogenic in situ hybridization (CISH) may be performed as an alternative to FISH. In CISH,
the HER2 probe is visualized by an immunoperoxidase reaction. This enables CISH results to
be scored using a conventional light microscope rather than the fluorescence microscope that
is required for FISH.
In contrast to the FISH assay, IHC assesses HER2 status at the protein level. The level of HER2
protein expression is scored on a semiquantitative scale. Tumors with 3+ protein expression are
scored as positive for HER2 protein expression, while tumors with 0 or 1+ protein expression
are scored as negative. Tumors with intermediate staining patterns (eg, cases showing complete
membrane staining that is weak in intensity) are considered equivocal; in these cases, FISH may
be performed to resolve HER2 status.
Multigene Prognostic Assays

Recently, clinical assays that utilize expression information gathered across a panel of genes
have been developed to predict recurrence risk and guide adjuvant chemotherapy decisions in
patients with early stage breast cancer. Several assays, which examine different sets of genes, are
currently available. Two of the more widely validated testing platforms involve examination of
21 and 70 genes, respectively. Depending on the particular testing platform, fresh frozen tissue
or formalin-fixed, paraffin-embedded tissue may be required. While methods used to quantify
gene expression vary by platform, 1 approach involves using the enzyme reverse transcrip-
tase to convert messenger RNA into complementary cDNA; the resulting cDNA then serves
as a template for assays such as quantitative polymerase chain reaction or microarray gene
expression profiling. While the role for these multigene prognostic assays in the routine clini-
cal management of patients with breast cancer remains to be fully established, several of these
assays have been validated in retrospective studies, and their utilities are now being examined
in prospective studies.
Serum-based Biomarkers in Breast Cancer

Serum-based tumor markers may be useful in the identification and management of patients
with breast cancer. The ideal breast cancer marker would be both specific for breast cancer and
sufficiently sensitive for screening purposes. Unfortunately, no marker identified to date meets
these criteria. However, some markers may have utility in evaluating the progression of disease
after initial therapy and for monitoring subsequent treatment.
When considering the use of serum tumor markers, several points should be kept in
mind: 1) none of the currently available markers is elevated in all patients with breast cancer,
The ideal breast cancer even in the setting of advanced disease, so that a normal tumor marker level does not exclude a
marker would be both malignancy; 2) these markers are most sensitive for detecting metastatic disease and have little
specific for breast cancer value in the diagnosis of local or regional recurrences; 3) the magnitude of change in marker levels
and sufficiently sensitive that correlates with disease progression or regression has not been firmly established; 4) tumor
for screening purposes. marker levels may paradoxically rise after initiation of chemotherapy, a phenomenon attributed to
Unfortunately, no marker therapy-mediated apoptosis or necrosis of tumor cells; 5) tumor marker levels may be increased in
identified to date meets the setting of certain benign diseases.
these criteria. However,
some markers may have
utility in evaluating the CA 15-3 and CA 27.29
progression of disease CA 15-3 and CA 27.29 represent different but overlapping epitopes of the MUC1 protein, a large,
after initial therapy complex glycoprotein expressed at the luminal surface of glandular epithelial cells. In malignant
and for monitoring cells, MUC1 may be overexpressed, and increased amounts of MUC1 may be shed into the circu-
subsequent treatment. lation. MUC1 levels in serum may be assessed by immunoassays employing distinct monoclonal
antibodies that recognize either the CA 15-3 or CA 27.29 epitopes. Results obtained from assays
assessing CA 15-3 and CA 27.29 are highly correlated. Importantly, CA 15-3 elevations have been
shown to occur in other malignancies and, to a lesser extent, in nonneoplastic disease. These
pathologic conditions include adenocarcinomas of the colon, lung, ovary, and pancreas, as well as
chronic hepatitis, cirrhosis, sarcoidosis, tuberculosis, and systemic lupus erythematosus.
In early stage breast cancer, elevated CA 15-3 levels are associated with a worse prognosis.
However, because CA 15-3 and CA 27.29 show fairly low sensitivity for detection of early disease,
the role of these markers in the management of early stage breast cancer remains unclear.
Measurement of either CA 15-3 or CA 27.29 is therefore not recommended for screening,
diagnosis, or staging of breast cancer. After primary and/or adjuvant therapy, increases in CA
15-3 or CA 27.29 can predict recurrence several months before other testing modalities or the
development of symptoms. However, because prospective, randomized clinical trials have yet to
demonstrate if such early detection of occult or asymptomatic metastases impacts disease-free
or overall survival, the routine use of CA 15-3 and CA 27.29 for this application is not currently
recommended. In patients with metastatic disease who are undergoing active therapy, CA 15-3 or
CA 27.29 testing may be used in conjunction with history, physical exam, and diagnostic imaging
to monitor the response to treatment. While the use of CA 15-3 or CA 27.29 alone to monitor
response to treatment is not recommended, in the absence of readily measurable disease, an
increasing CA 15-3 or CA 27.29 may be used nonetheless to identify treatment failure. In most
clinical trials to date, a significant alteration in CA 15-3 has been defined as a concentration
change of at least 25%.
Carcinoembryonic Antigen (CEA)

CEA is a cell-surface glycoprotein involved in cell adhesion that is normally expressed in the
developing fetus. CEA levels in the blood decrease to very low levels after birth, although levels
may be elevated slightly in smokers. CEA expression may be elevated in several types of cancer,
including cancers of the breast, colon, pancreas, lung, and ovary, because of antigen shedding
into the circulation. CEA may also be elevated in nonneoplastic diseases, including inflammatory
bowel disease, pancreatitis, and liver disease. CEA is detected by immunoassay.
In early stage breast cancer, elevated CEA levels are associated with a worse prognosis. CEA
is not recommended for screening, diagnosis, staging, or routine surveillance of breast cancer
patients after primary therapy. Like CA 15-3 and CA 27.29, CEA testing may be used in conjunc-
tion with history, physical exam, and diagnostic imaging to monitor the response to treatment in
patients with metastatic disease who are undergoing active therapy. However, CEA should not be
used alone for this purpose. Compared with CA 15-3, CEA is generally a less sensitive marker for
breast cancer. However, in some patients with breast cancer, elevations of CEA may occur in the
setting of normal CA 15-3 or CA 27.29 levels.
HEREDITARY BREAST AND OVARIAN CANCER SYNDROME

Description
While most cases of breast cancer are caused by acquired somatic mutations, approximately 5%
to 10% of breast cancer cases are attributed to a germline mutation in a highly penetrant cancer
predisposition gene. A large proportion of these hereditary cases are associated with mutations
in 2 genes, BRCA1 and BRCA2. Mutations in BRCA1 and BRCA2 cause the hereditary breast Approximately 5% to
and ovarian cancer syndrome, an autosomal dominant disorder in which the risk of both breast 10% of breast cancer
and ovarian cancer is significantly increased compared with the general population. BRCA1 and cases are attributed to a
BRCA2, which are located at chromosome 17q21 and 13q12, respectively, are tumor suppres- germline mutation in a
sor genes that play essential roles in the repair of double-stranded DNA breaks and thus in the highly penetrant cancer
maintenance of genome stability. Accordingly, in tumors from individuals with hereditary breast predisposition gene.
and ovarian cancer syndrome, the wild-type BRCA1 or BRCA2 allele is deleted, consistent with a A large proportion of
tumor suppressor function for BRCA1 and BRCA2. these hereditary cases
are associated with
A woman’s risk for harboring either a BRCA1 or BRCA2 mutation is increased by certain
mutations in 2 genes,
factors related to her personal and family medical history. Personal factors include: 1) early onset
BRCA1 and BRCA2.
breast cancer (ie, diagnosis before 50 years of age); 2) bilateral or multifocal breast cancers; and
3) a history of both breast and ovarian cancer. Factors from the family history include: 1) breast
cancer or breast and ovarian cancer in a pattern consistent with autosomal dominant transmis-
sion; and 2) breast cancer in a male relative.
Hereditary breast and ovarian cancer syndrome shows incomplete penetrance. Among
women with either a BRCA1 or BRCA2 mutation, the lifetime risk of developing breast cancer is
60% to 80%. The lifetime risk of developing ovarian cancer is 15% to 60% for women with BRCA1
mutations and 10% to 27% for women with BRCA2 mutations. Mutations in BRCA1 and BRCA2
also increase the risk of male breast cancer. Individuals with hereditary breast and ovarian can-
cer syndrome are also at risk for other tumors, including melanoma and cancers of the prostate
(in males) and pancreas.
Determination of BRCA1 and BRCA2 mutation status is an important clinical assessment, as
the identification of a deleterious mutation may alter clinical management. Interventions avail-
able to BRCA1 and BRCA2 mutation carriers include intensive screening, chemoprevention,
prophylactic mastectomy, and prophylactic oophorectomy. Prophylactic oophorectomy, which
reduces the risk of breast cancer as well as ovarian cancer, is recommended for all mutation carri-
ers at the completion of childbearing.
Determination of BRCA1 and BRCA2 Mutations

Genetic testing for BRCA1 and BRCA2 mutations should be offered to individuals with a personal
or family history suspicious for hereditary breast and ovarian cancer syndrome, and to women at
risk because of a family member known to harbor a deleterious mutation in 1 of these genes. It is
Genetic testing for BRCA1
critical that testing be offered in the setting of appropriate genetic counseling, so that individuals
and BRCA2 mutations
are provided appropriate information regarding the risks, benefits, and limitations of genetic test-
should be offered to
ing. Such counseling should also include consideration of how the results of such testing might
individuals with a
affect other family members. personal or family history
Study of kindreds with hereditary breast and ovarian cancer syndrome has identified suspicious for hereditary
hundreds of different deleterious mutations in the BRCA1 and BRCA2 genes. Most consist breast and ovarian cancer
of frameshift or nonsense mutations, which are predicted to result in a loss of function of the syndrome, and to women
encoded gene product. Due to the large number of different mutations described, genetic test- at risk because of a family
ing to assess for most of these involves examination of the DNA sequence of the entire coding member known to harbor
region of each gene. Additional molecular testing may also be employed to assess for certain a deleterious mutation in
large genomic rearrangements that cannot be detected by routine DNA sequencing. In cases 1 of these genes.
where a known deleterious mutation has already been identified in a family member, targeted
mutation analysis is performed to specifically assess for the familial mutation.
When possible, genetic testing should be performed on an individual who has been diag-
nosed with breast or ovarian cancer because this strategy provides the most information for other
members of the family. Genetic testing can lead to 4 possible outcomes: a true-positive result,
a true-negative result, an uninformative result, and a variant of uncertain significance. A true-
positive result occurs when a deleterious mutation known to be associated with increased cancer
risk is identified; such a result confirms the diagnosis of hereditary breast and ovarian cancer
syndrome. A true-negative result occurs only when the tested individual is found to lack a specific
deleterious mutation already known to run in the family; a true-negative result thus reduces the
tested individual’s risk of developing breast and/or ovarian cancer to that of general population.
An uninformative result occurs when a mutation is not identified in an individual from a fam-
ily in which a deleterious mutation has not yet been identified; an uninformative result does not
exclude the possibility of a BRCA1 or BRCA2 mutation that cannot be detected by current testing
methodologies, nor does it exclude the possibility of a mutation in another cancer susceptibility
gene. Genetic variants of uncertain significance are typically missense variants of unknown func-
tional significance or intronic variants not predicted to disrupt mRNA processing; individuals
harboring variants of unknown significance may still be at increased risk for cancer, and their
medical management should be based on the known family history. In all cases, posttest genetic
counseling should be performed to ensure that individuals fully comprehend the implications of
their testing results.
OTHER HIGHPENETRANCE CANCER PREDISPOSITION GENES

In addition to BRCA1 and BRCA2, highly penetrant forms of hereditary breast cancer have also
been linked to mutations in the tumor suppressor genes TP53, PTEN (phosphatase tensin homo-
log), and STK11. While mutations in these genes comprise a smaller proportion of hereditary
breast cancer cases than BRCA1 and BRCA2, identification of these mutations similarly has pro-
In addition to BRCA1 and found implications for clinical management and for genetic risks of family members. Genetic
BRCA2, highly penetrant testing for mutations in these genes is conducted using molecular approaches similar to those
forms of hereditary breast used for BRCA1 and BRCA2 and can also lead to the same 4 possible test outcomes described
cancer have also been above. Genetic testing for these mutations should be offered in the setting of appropriate genetic
linked to mutations in the counseling to at-risk family members of an individual with a known deleterious mutation and to
tumor suppressor genes individuals with personal or family histories suspicious for these particular syndromes, which are
TP53, PTEN (phosphatase described in detailed below.
tensin homolog), and
STK11.
Li-Fraumeni Syndrome
Germline mutations in TP53 cause Li-Fraumeni syndrome, a rare autosomal dominant condi-
tion associated with the development of a variety of tumor types throughout life, including in
childhood. TP53, located at chromosome 17p13, encodes the p53 protein, which plays key roles
in DNA repair, cell cycle control, and the initiation of apoptosis. In addition to breast cancer,
tumors associated with Li-Fraumeni syndrome include osteosarcomas, soft tissue sarcomas, brain
tumors, leukemias, and adrenocortical carcinomas. In families with Li-Fraumeni syndrome,
breast cancer is common and may account for one third of all cancers.
Cowden Syndrome
Germline mutations in PTEN cause Cowden syndrome, a rare autosomal dominant condi-
tion characterized by an increased risk of certain cancers and the development of multiple skin
and mucosal hamartomas (focal malformations that resemble neoplasms but result from faulty
development of the tissue). PTEN, located at chromosome 10q23, encodes a phosphatase that
downregulates the phosphatidylinositol-3-kinase (PI3K) signal transduction pathway, thereby
contributing to the regulation of cell growth. The spectrum of cancers seen in Cowden syndrome
includes thyroid cancer, uterine cancer, renal cell cancer, and breast cancer. Females with Cowden
syndrome have a 25% to 50% lifetime risk of developing breast cancer.
Peutz-Jeghers Syndrome
Germline mutations in STK11 cause Peutz-Jeghers syndrome, a rare autosomal dominant condi-
tion characterized by gastrointestinal polyps and mucocutaneous pigmentation. STK11, located
at chromosome 19p13, encodes serine/threonine-protein kinase 11 and functions to regulate
cell polarity, energy utilization, and apoptosis. Individuals with Peutz-Jeghers syndrome are at
increased risk for the development of a variety of cancers, including colorectal, gastric, pancreatic,
ovarian, and breast. The lifetime risk of breast cancer reported for females with Peutz-Jeghers
syndrome has been estimated at 30% to 50%.
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C H A P T E R
The Endocrine System

Alison Woodworth, Vipul Lakhani, Samir L. Aleryani,
and Michael Laposata 22
LEARNING OBJECTIVES
1. Learn the physiology and biochemistry of the relevant hormones and other
important mediators.
2. Understand the laboratory tests used in the diagnosis of the more commonly
encountered disorders.
3. Identify the clinical disorders associated with each of the endocrine glands
and the role of specific laboratory tests in their diagnosis.
CHAPTER OUTLINE
Introduction 422 Adrenal Medulla 441
Thyroid 422 Physiology and Biochemistry 441
Physiology and Biochemistry 422 Laboratory Tests 441
Laboratory Tests 422 Pheochromocytoma 442
Hyperthyroidism Overview and Parathyroid Glands 444
Associated Disorders 425 Physiology and Biochemistry 444
Hypothyroidism Overview and Laboratory Tests 445
Associated Disorders 428 Primary Hyperparathyroidism 447
Pregnancy-related Thyroid Disease 429 Secondary Hyperparathyroidism 448
Euthyroid Sick Syndrome 429 Hypoparathyroidism 448
Thyroid Tumors 429 Pseudohypoparathyroidism 449
Adrenal Cortex 430 Vitamin D Deficiency 449
Physiology and Biochemistry 430 Hypercalcemia of Malignancy 449
Laboratory Tests 432 Hypocalciuric Hypercalcemia 450
Hyperfunction Involving Osteoporosis 450
Glucocorticoids With or Without Osteomalacia 450
Mineralocorticoids: Cushing Osteitis Deformans (Also Known as Paget
Syndrome 434 Disease of Bone) 451
Hypofunction Involving Glucocorticoids Testes and Ovaries 451
With or Without Mineralocorticoids: Male Physiology and Biochemistry 451
Adrenal Insufficiency 436 Female Physiology and Biochemistry 454
Hyperfunction and Hypofunction Disorders Related to the Pituitary Gland 457
Involving Mineralocorticoids: Growth Hormone/Anterior Pituitary 457
Hyperaldosteronism and Prolactin/Anterior Pituitary 460
Hypoaldosteronism 438 Antidiuretic Hormone (ADH)/Posterior
Alterations in the Synthesis of Pituitary 461
Glucocorticoids, Mineralocorticoids, Neoplastic Disorders 464
and Sex Steroids: Congenital Adrenal Multiple Endocrine Neoplasia 464
Hyperplasia 440 Carcinoid Tumors 465
421
Patient presents with signs and

symptoms of an endocrine disorder
Identify the endocrine gland(s) affected

and determine whether there is excess or
inadequate hormone secretion from the
gland(s) or an ectopic source
Determine the precise cause of the

abnormality to direct treatment appropriately
FIGURE 22–1 An approach to the patient with an endocrinologic disorder.
INTRODUCTION
This chapter on endocrine disorders is divided into separate discussions of each of the endocrine
glands. Each section begins with an overview of the physiology and biochemistry of the relevant
hormones. In addition, because of the large number of (often complex) laboratory tests in endo-
crinology, each section has a brief description of the laboratory tests most frequently used to diag-
nose the disorders in that disease group. Tests for which either serum or plasma is an acceptable
specimen for analysis are noted as serum tests. Tests specifically requiring plasma are indicated
by inclusion of the word “plasma” before the test name. As with all other chapters, each disorder
is presented with a description of the disease and information useful in establishing a diagnosis.
Figure 22–1 shows a general approach to the patient with endocrine disease.
THYROID
Physiology and Biochemistry
Production of thyroid hormones is regulated by the hypothalamic–pituitary–thyroid axis
(Figure 22–2). Thyrotropin-releasing hormone (TRH) is produced in the hypothalamus and
A “generational” induces thyroid-stimulating hormone (TSH or thyrotropin) production in the anterior pituitary.
classification has TSH, in turn, stimulates thyroid hormone production and release by the thyroid gland. TSH pro-
been applied for TSH duction is inversely related to plasma thyroxine (T4) and triiodothyronine (T3) concentrations.
assays based on the The 2 primary hormones synthesized and secreted by the thyroid gland are T4 and, in lesser quan-
assay sensitivity. Third- tities, T3 (Figure 22–3). They are transported by plasma proteins—notably thyroid-binding globu-
generation assays can lin (TBG), transthyretin, and albumin—to various tissue sites where T4 is deiodinated to the active
accurately measure TSH form, T3, and the inactive form known as reverse T3 (rT3). Thyroid hormones act through nuclear
as low as 0.01 mU/L. hormone receptors that are transcription factors for numerous genes. These genes regulate a num-
ber of critical physiologic functions in development and metabolism.
Laboratory Tests
TSH
A “generational” classification has been applied for TSH immunoassays based on the assay sensi-
tivity. Third-generation assays can accurately measure TSH as low as 0.01 mU/L. This allows the
physician to distinguish mildly subnormal TSH values from the low values of overt hyperthyroid
patients. The third-generation tests are also useful for evaluating the effectiveness of the thyroid
[–]
Hypothalamus
Thyrotropin-releasing
hormone (TRH)
[+]
[–]
Pituitary
T3
Thyroid-stimulating
hormone (TSH) T4
[+] rT3
T3 is primary
Thyroid feedback stimulus
FIGURE 22–2 Hypothalamic–pituitary–thyroid interactions. [+] Stimulation; [−] inhibition.
hormone replacement in hypothyroid patients. Third-generation assays are essential for monitor-
ing TSH suppression therapy in patients with a TSH-responsive thyroid tumor.
The relationship between TSH and the thyroid hormones, particularly free T4, is an inverse
log-linear one, such that very small changes in free T4 result in large changes in TSH. Thus, TSH
is the most sensitive first-line screening test for suspected thyroid abnormalities. If the TSH is
within the normal reference range, no further testing is performed. If the TSH is outside of the
reference range, a free T4 is obtained.
Uptake into cells

Oral intake Plasma producing thyroid homone Thyroidal
of iodine iodide iodide
Binding of iodide
to thyroglobulin
Coupling of Monoiodotyrosine
Proteases Thyroglobulin
iodotyrosines (MIT) and
Free T4 release T3 bound diiodotyrosine
and T4 from T3 and T4 (DIT) residues on
thyroglobulin
thyroglobulin
Deiodination
Free T3 Thyroid hormones

transported to tissues
on thyroid-binding
globulin (TBG), transthyretin,
Reverse and albumin Diverse metabolic
T3 (rT3) effects on target cells
Free T4
FIGURE 22–3 The formation, secretion, and transport of thyroid hormones.

Total Thyroid Hormone Measurements

Assays are available for both total T4 and total T3 measurements. These assays are quite specific
and suffer little interference. However, transient changes in serum thyroid hormone-binding pro-
tein concentrations may affect total T4 and T3 concentrations. Therefore, an assessment for free
T4 (see below) is usually more helpful in evaluating thyroid function. The concentration of T4 in
the blood is usually 100 to 200 times greater than the T3 level. In hyperthyroidism, total T3 and
T4 concentrations correlate in all but a small subset of patients who have an elevation only in T3.
For that reason, T3 should be measured in the serum of patients clinically suspected to be hyper-
thyroid and who have normal concentrations of serum T4. Measurement of T3 concentrations has
limited clinical utility in assessment of hypothyroidism. Significant decreases in total T3 are seen
in euthyroid sick syndrome (ETS).
Free Thyroid Hormones and Thyroid Hormone-binding Capacity

“Direct” free thyroid hormone assays, without the need for a preliminary step to separate free
hormones from hormones bound to protein carriers, are available for measurement of free T4 and
free T3. Only a small fraction of T4 (<0.1%) circulates unbound to proteins, and this has made the
accurate quantitation of free T4 analytically difficult. Free T4 is a better indicator of thyroid status
than total T4 because, as noted above, the total T4 is altered by changes in the amounts of TBG,
albumin, and other thyroid hormone-binding proteins. About 0.3% of T3 circulates as free T3. In
general, free T3 concentrations correlate well with total T3, but as with T4, the concentrations of
thyroid hormone-binding proteins influence the total T3 level.
Although the free T4 index offers an approximation of free T4, this measure is largely obsolete
due to improvements in the free hormone assays. The free T4 index is calculated by multiplying the
total T4 by the measure of available hormone-binding sites on TBG in an assay known as the percent
T3 uptake. Because of the dependence on the amount of TBG, the free T4 index and the T3 uptake
Antithyroid antibodies are are affected by changes in the amount of thyroid-binding proteins induced by a variety of stimuli.
present in approximately
15% of the general
Reverse Triiodothyronine
population and are the
most common cause of Under acute stress or in illness, there is a shift in the T4 deiodination in favor of the inactive rT3
thyroid disease in iodine- form rather than the active T3. Numerous immunoassays are available for the measurement of
replete societies. rT3 using antisera that do not cross-react significantly with T3 or T4. rT3 is markedly increased in
ETS syndrome (see below), but its measurement is rarely required for this diagnosis because its
increase is proportional to the decrease in T3.
Antithyroid Antibodies
Antithyroid antibodies are present in approximately 15% of the general population and are the
most common cause of thyroid disease in iodine-replete societies. They are also present in selected
autoimmune diseases not usually associated with thyroid dysfunction. Descriptions of 3 types of
antithyroid antibodies follow:
• Antimicrosomal/antithyroid peroxidase antibodies (anti-TPO)—These antibodies are
directed against a protein component of thyroid microsomes, the enzyme TPO. They are
present in almost all patients with Hashimoto thyroiditis, in about 85% of patients with
Graves disease (both discussed below), and in some patients with type 1 insulin-dependent
diabetes mellitus, celiac disease, and Addison disease. An elevated titer of anti-TPO anti-
bodies in the context of clinical symptoms of thyroid dysfunction and abnormal TSH and
free T4 results is diagnostic for autoimmune thyroid disease. The presence of TPO antibod-
ies before or during pregnancy is a good predictor of those women who will develop post-
partum thyroid disease (discussed below). Normal concentrations are not well established
because the antibodies may be found in healthy people (up to 12% of the population), and
the reference range depends on the method used to perform the test.
• Antithyroglobulin antibodies—These are also called colloidal antibodies. They are present
in more than 85% of patients with Hashimoto thyroiditis and in more than 30% of patients
with Graves disease. Like anti-TPO, antithyroglobulin antibodies also may be found in
other autoimmune diseases. In iodine-sufficient areas, the antithyroglobulin antibody test is
used less often, in favor of anti-TPO. However, in patients with suspected iodine deficiency,
antithyroglobulin antibody is a better indicator of autoimmune thyroid disease.
• TSH receptor antibodies—These are a diverse group of immunoglobulins that bind to TSH
receptors and influence their action. They are found in most patients with Graves dis-
ease and in patients with selected other autoimmune disorders involving the thyroid. The
biological functions of these antibodies vary from thyroid stimulation to thyroid inhibition
(by blocking stimulation induced by TSH). Antibodies referred to as thyroid-stimulating
immunoglobulins are present in 95% of patients with untreated Graves disease. In vitro
bioassays can assess the ability of stimulatory antibodies to induce functional responses in
cultured cells by measuring cyclic adenosine monophosphate increases or adenylate cyclase
activity. Assays are available that measure the capability of the inhibitory antibodies, called
thyrotropin-binding inhibitory immunoglobulins, to block the binding of labeled TSH to
its receptors.
Radioactive Iodine Uptake and Thyroid Scans

The radioactive iodine uptake and thyroid scan measurements involve the in vivo administration
of radioactive iodine. Accumulated radioactivity in the thyroid is measured at intervals within
24 hours using a gamma scintillation counter. A nuclear imaging scan that examines the ana-
tomic distribution of radioactive iodine uptake within the thyroid gland also may be obtained.
Radioactive iodine uptake studies may be helpful when the diagnosis is in question and in dif-
ferentiating between possible causes of hyperthyroidism.
Thyroglobulin
Thyroglobulin is stored in the follicular colloid of the thyroid as a prohormone. Thyroglobulin
measurements are used to monitor treatment in thyroid cancer. Persistent serum thyroglobulin
after thyroidectomy is evidence of incomplete ablation or metastatic thyroid cancer. Thyroglobu-
lin concentrations should always be assessed in the context of an antithyroglobulin antibody test
because these autoantibodies can cause false-positive or -negative thyroglobulin results.
Calcitonin
Calcitonin is a polypeptide hormone produced by the thyroidal C cells, whose primary function
is in regulating calcium homeostasis. It is elevated in patients with C-cell hyperplasia and medul-
lary thyroid carcinoma (MTC). Calcitonin measurements are used to determine when to perform
prophylactic thyroidectomy on patients with familial MTC. In addition, it is used to assess prog-
nosis and monitor recurrence of MTC post thyroidectomy.
Fine Needle Aspiration (FNA) Cytology

FNA is the procedure of choice to collect a specimen for microscopic review to distinguish benign In the presence of a
from malignant thyroid nodules. The sensitivity of thyroid FNA for detection of thyroid cancer clinical history and
and other disorders varies from 70% to 97%. It depends greatly on the quality of the specimens physical examination
and the experience of the cytopathologist. Thyroglobulin can also be measured in lymph node consistent with
FNA aspirates to diagnose and monitor thyroid cancer. hyperthyroidism,
a diagnosis of
hyperthyroidism (but
Hyperthyroidism Overview and Associated Disorders not necessarily its cause)
Description and Diagnosis can be established by
the demonstration of a
Hyperthyroidism, also known as thyrotoxicosis, is a collection of disorders associated with excess low TSH level and a high
thyroid hormone (Table 22–1). There are 4 main causes of hyperthyroidism: 1) overstimulation free T4.
of the thyroid (elevated TSH, human chorionic gonadotropin [hCG], and/or TSH receptor auto-
antibodies [TRAbs]); 2) genetic mutations leading to increased synthesis and secretion of thy-
roid hormone (germline, sporadic, or tumor induced); 3) release of excess hormone from the
thyroid (inflammation, infection, injury); 4) extrathyroidal sources of thyroid hormone (ectopic
thyroid tissue or exogenous hormone). Patients with hyperthyroidism demonstrate a spectrum of
hypermetabolic features, including nervousness, palpitations, muscle weakness, increased appe-
tite, diarrhea, heat intolerance, warm skin, weight loss, and perspiration. Affected patients also
TABLE 22–1 Laboratory Evaluation of Patients for Thyroid Disease

Laboratory Test Results Suggestive of Diagnosis in the Appropriate
Disorder Clinical Setting
Hyperthyroidism
Graves disease TSH low; free T4 high; in some cases, T3 is elevated and free T4 is normal;
TRAbs or TSI elevated
Toxic multinodular goiter TSH low; free T4 and T3 normal or high; normal or increased radioactive iodine
uptake; thyroid scan with multiple areas of increased uptake surrounded by
suppressed uptake
Toxic adenoma TSH low; free T4 and T3 normal or high; normal or increased radioactive iodine
uptake; thyroid scan with focal increased uptake in tumor surrounded by
suppressed uptake in nontumor tissue
Subacute thyroiditis TSH low; free T4 and T3 high; increased; decreased radioactive iodine uptake
Painless thyroiditis TSH low; free T4 and T3 high; erythrocyte sedimentation rate normal;
decreased radioactive iodine uptake
Hypothyroidism
Hashimoto thyroiditis TSH high; T4 normal and then low, preceding a decline in T3; anti-TPO and/or
antithyroglobulin antibody positive
Ablative hypothyroidism TSH high; free T4 and T3 low following procedure that ablates thyroid
Infantile hypothyroidism TSH high; free T4 low in a newborn or infant
Euthyroid sick syndrome TSH normal to high; free T4 normal; T3 low; rT3 high; concentrations of TSH and
thyroid hormones vary throughout disease course
rT3, reverse triiodothyronine; T3, triiodothyronine; free T4, free thyroxine; TSH, thyroid-stimulating hormone; TRAbs, TSH receptor
autoantibodies; TSI, thyroid-stimulating immunoglobulins.
may have exophthalmos, emotional changes, menstrual changes, and a fine tremor of the hands.
In the presence of a clinical history and physical examination consistent with hyperthyroidism,
a diagnosis of hyperthyroidism (but not necessarily its cause) can be established by the demon-
stration of a low TSH level and a high free T4. In uncommon situations, only the total T3 level
is elevated and the serum free T4 is normal (T3 thyrotoxicosis). To determine the etiology of the
hyperthyroidism, additional testing is usually necessary. Graves disease, toxic multinodular goiter
(TMNG), and toxic adenoma account for the vast majority (>95%) of cases of hyperthyroidism. It
should be noted that diffuse or focal enlargement of the thyroid gland, also known as goiter, can
be associated with hyperfunction, normal function, and hypofunction of the gland.
Thyroid Storm
Thyroid storm is a relatively uncommon, but life-threatening manifestation of hyperthyroid-
ism caused by excess circulation of thyroid hormones. Symptoms of thyroid storm are similar,
Graves disease is a but much more severe than traditional hyperthyroidism, including a markedly high fever of
relatively common 105°F to 106°F, tachycardia, hypertension, and neurological and gastrointestinal abnormali-
hyperthyroid disorder ties. Thyroid storm is precipitated by acute illnesses such as sepsis, diabetic ketoacidosis, and
occurring more preeclampsia, as well as surgical or other diagnostic or therapeutic actions such as radioac-
frequently in women. tive iodine use, anesthesia, excessive thyroid hormone ingestion, or thyroid palpation. Thyroid
It is an autoimmune storm is associated with a high fatality rate if not identified early. The diagnosis is based on the
disease caused by TSH presence of clinical signs and symptoms of severe hyperthyroidism in the context of a precipi-
receptor autoantibodies tating cause. In addition, marked elevations in free and total T4 are common in thyroid storm.
that bind to and stimulate Total T3 is unreliable in this setting because concomitant nonthyroidal illness (NTI) may cause
TSH receptors resulting in
T3 to decrease significantly.
autonomous production
of thyroid hormone.
Graves Disease
Graves disease is a relatively common hyperthyroid disorder occurring more frequently in
women. It has a familial predisposition. It is an autoimmune disease caused by TSH receptor
antibodies (TRAbs) that bind to and stimulate TSH receptors resulting in autonomous produc-
tion of thyroid hormone. While many patients have the classic signs and symptoms of thyro-
toxicosis, in elderly patients with Graves disease, apathy, muscle weakness, and cardiovascular
abnormalities occur more often than hypermetabolic symptoms.
Laboratory tests show undetectable TSH and increased free T4. In some cases, the T3 is ele-
vated and the T4 is normal. The differential diagnosis includes TMNG, toxic adenoma, painless
and subacute thyroiditis, ectopic thyroid tissue, and anxiety states (see below for descriptions).
Detection of TRAbs along with the results from radioactive iodine uptake and nuclear thyroid
scans are helpful in distinguishing among these possibilities. There is usually increased radioac-
tive iodine uptake in Graves disease. The pattern on imaging is diffuse.
Toxic Multinodular Goiter

The cause of hyperthyroidism in patients with TMNG is an apparent functional autonomy of cer-
tain areas within the thyroid gland. The disorder is seen more commonly in elderly patients. The
degree of hyperthyroidism is generally less severe than that found in Graves disease. Cardiovas-
cular symptoms are prominent, such as arrhythmias, atrial fibrillation, or congestive heart failure,
with weakness and wasting.
Laboratory tests usually show low or undetectable TSH and normal or elevated free T4 and T3
concentrations and no evidence of thyroid autoantibodies. Patients with TMNG will have normal
to high radioactive iodine uptake, and the thyroid scan shows iodine localized to active nodules.
Toxic Adenoma
Thyroid adenomas that secrete thyroid hormones and cause hyperthyroidism are known as toxic
adenomas. Thyroid hormone synthesis by a toxic adenoma is usually independent of TSH regula-
tion, and it results in suppression of TSH secretion. These tumors can usually be distinguished
from TMNG and Graves disease by a radioactive iodine uptake study and thyroid scan because
there is localized uptake in the adenoma and little or no uptake in surrounding thyroid tissue.
Thyroiditis
Subacute Thyroiditis. Subacute thyroiditis is produced by a viral infection that alters thyroid
function. This disease usually lasts for months, with thyroid function eventually returning to nor-
mal. The patient often has an associated upper respiratory infection, fever, and local pain mimick-
ing a sore throat or an earache.
Patients in the early stage of this disease may have hyperthyroidism, with elevated T4 and T3
concentrations and a low TSH. Laboratory findings also often include a high erythrocyte sedi-
mentation rate and little to no radioactive iodine uptake. If the disease progresses and the thyroid
hormones are depleted, the patient develops hypothyroidism with low T3 and T4 concentrations
and an elevated TSH.
Postpartum Thyroiditis. Postpartum thyroid disease is a transient inflammatory process that
has an onset of 1 to 6 months postpartum. Although the etiology is unclear, it is thought to be
caused by a rebound in the immune system in response to the general state of immunosuppres-
sion that occurs during pregnancy. This disease can present as either hyperthyroidism or hypo-
thyroidism. The typical disease course begins with a period of hyperthyroidism with elevated free When hypothyroidism
T4, reduced TSH, and little to no radioactive iodine uptake for 3 to 6 months. This is followed occurs during
by a 3- to 6-month period of hypothyroidism associated with reduced concentrations of free development and in
T4 and elevated TSH that completely resolves after 1 year. Approximately 20% of women with infancy, it results in
postpartum hypothyroidism develop permanent disease, requiring lifelong treatment and moni- a condition known
toring. The presence of anti-TPO antibodies prior to and during pregnancy is associated with an as cretinism, which is
increased risk for postpartum thyroiditis. marked by retardation
of physical and
Painless Thyroiditis. Painless thyroiditis may be induced by numerous drugs including lithium, intellectual growth. When
interferon, and in a small portion of patients on amiodarone therapy. Further, some patients with hypothyroidism first
chronic thyroiditis have a transient painless thyrotoxicosis, of unclear etiology. appears in older children
Typically, free T4 and T3 concentrations are elevated with a low TSH. Patients have a mark- and adults, the collection
edly depressed radioactive iodine uptake. Painless thyroiditis can be distinguished clinically from of signs and symptoms is
subacute thyroiditis because an elevated erythrocyte sedimentation rate and local pain in the known as myxedema.
region of the thyroid are more consistent with subacute thyroiditis. A definitive diagnosis can be
made by microscopic review of cells obtained by aspiration or biopsy. Thyroglobulin measure-
ments can differentiate among patients with chronic thyroiditis from those with thyrotoxicosis
caused by surreptitious thyroid hormone intake.
Hypothyroidism Overview and Associated Disorders

When hypothyroidism occurs during development and in infancy, it results in a condition
known as cretinism, which is marked by retardation of physical and intellectual growth. In
95% of cases, hypothyroidism originates in the thyroid gland itself. If a patient has an increased
serum TSH and a decreased free T4—together with appropriate clinical symptoms—a diagnosis
of hypothyroidism is confirmed (Table 22–1). In asymptomatic patients, increased TSH, accom-
panied by a normal free T4, is known as subclinical hypothyroidism and may be indicative of
early stages of primary hypothyroidism. High titers of anti-TPO antibodies suggest Hashimoto
thyroiditis (see below) or postpartum thyroid dysfunction in a postpartum woman. While in
the United States, autoimmunity is the main cause of hypothyroidism, iodine deficiency is the
primary cause worldwide.
Hypothyroidism also may be a result of inadequate stimulation of the thyroid by TSH. This
is known as secondary hypothyroidism. A subnormal free T4 with a decreased or inappropriately
normal TSH is suggestive of secondary hypothyroidism from decreased TSH production or pro-
duction of a biologically inactive form of TSH in the pituitary. It is usually accompanied by other
pituitary hormone deficiencies, and it is much less common than primary hypothyroidism.
Clinical pictures of hypothyroidism differ, depending on the age. Congenital hypothyroidism
is characterized by low production of thyroid hormones and can result in growth and intellectual
delay if untreated. In the United States, all states screen for congenital hypothyroidism by testing
for elevated TSH or a combination of elevated TSH and decreased free T4. Significant changes in
thyroid function occur in the neonatal period and throughout childhood. Therefore, TSH and
free T4 concentrations should be assessed using age-specific reference intervals. In particular, T4
is typically elevated in newborns. In adults, hypothyroidism can have an insidious onset, espe-
cially in the elderly. Symptoms are usually nonspecific in the early stage and then progress to
more definitive characteristics of hypothyroidism with dry hair, dry skin, periorbital puffiness,
dull expression, large tongue, and enlarged heart. If untreated, myxedema coma with respiratory
failure may occur. Treatment involves hormone replacement.
Hashimoto Thyroiditis
Hashimoto thyroiditis is a common chronic inflammatory disease of the thyroid that accounts for
as many as 90% of all cases of hypothyroidism in areas of iodine sufficiency. Autoimmune factors
are thought to be the cause. Hashimoto thyroiditis is often associated with other autoimmune
diseases such as Sjögren syndrome and pernicious anemia.
Hashimoto thyroiditis Patients with Hashimoto thyroiditis carry anti-TPO and antithyroglobulin antibodies. Firm
is a common chronic thyroid enlargement and goiter is characteristic, but atrophy is also seen. Patients typically have
inflammatory disease of an increased TSH and may have a normal free T4 and an elevated radioactive iodine uptake in the
the thyroid that accounts early stage of the disease. Over time, serum T4 declines first, followed by a decline in T3 as hypo-
for as many as 90% of all thyroid symptoms become predominant.
cases of hypothyroidism.
Patients with Hashimoto Postablative Hypothyroidism
thyroiditis carry anti-TPO Postablative hypothyroidism is a relatively common cause of hypothyroidism in adults. Thyroid
and antithyroglobulin
ablation occurs with total or subtotal thyroidectomy, or following treatment with radioactive
antibodies.
iodine for hyperthyroidism.
A history of ablative therapy along with an elevated TSH and a low free T4 concentration
indicates that ablation has produced a hypothyroid state.
Infantile Hypothyroidism
Severe hypothyroidism in infancy is known as cretinism and, as previously noted, is charac-
terized by irreversible mental retardation and growth impairment unless treated promptly.
The appearance of symptoms depends on the severity of the disorder. However, even severe
hypothyroidism is not usually apparent at birth. Early diagnosis and treatment with thyroid hor-
mone prevents the manifestations of the disease. Elevated TSH and a low T4 in a newborn or
young infant are indicative of infantile hypothyroidism.
Pregnancy-related Thyroid Disease

Normal pregnancy is associated with a number of physiologic changes in thyroid function result-
ing in differences in “normal” laboratory values for thyroid function tests. The increase in estro-
gen stimulates hepatic synthesis of TBG, resulting in a net increase in total T3 and total T4 by
about 1.5-fold. Significant homology exists between hCG, the pregnancy-associated glycoprotein
hormone (see Chapter 20), and TSH. Because of this, hCG can directly stimulate the thyroid to
produce thyroid hormone. Excess production of thyroid hormone signals a downregulation of
TSH secretion. In the first trimester of pregnancy, increasing hCG concentrations are directly
mirrored by decreasing TSH concentrations, which return to low normal in the second and third
trimesters. Thus, TSH measurements in pregnancy should be considered in the context of gesta-
tional age and a reduced upper limit of normal. Many laboratories now report TSH with trimes-
ter-specific reference intervals.
Thyroid dysfunction during pregnancy can result in increased risks for the mother and fetus.
Hypothyroidism during pregnancy is associated with an increased risk of miscarriage or preterm
delivery and impaired neurological development in the fetus. Although controversial, it is recom-
mended to screen all high-risk and symptomatic pregnant women for hypothyroidism by mea-
suring TSH. Hyperthyroidism in pregnancy is associated with an increased risk of spontaneous
abortion, preterm delivery, preeclampsia, and thyroid anomalies in the newborn. A subnormal
TSH test result should be followed with free T4 testing. An elevated free T4 with the presence of
autoantibodies confirms the diagnosis of hyperthyroidism in pregnancy. In mothers with con-
firmed thyroid disease, fetal thyroid function can be evaluated with ultrasound and amniotic
fluid testing for TSH, free T4, and total T4. Normal reference intervals are instrument-specific for
amniotic fluid thyroid function tests.
Euthyroid Sick Syndrome

Description
It is estimated that 40% of emergency department patients have euthyroid sick syndrome (ETS)
at presentation. Stress, trauma, and illness can alter thyroid hormone production, transport, and
metabolism, and thereby TSH levels, because of disruption of the normal feedback relationship
between TSH and T3 and T4. This condition with altered thyroid hormone levels and no intrinsic The condition with
disorder of the thyroid gland is called ETS or NTI, of which there are several variants. altered thyroid hormone
levels and no intrinsic
Diagnosis disorder of the thyroid
gland is called euthyroid
There is no consensus in the literature regarding the diagnosis and also therapy of ETS. The cause
sick syndrome (ETS) or
of ETS is different from patient to patient and is dependent on the history and any endocrinologic nonthyroidal illness (NTI),
diagnosis. In moderately ill patients with ETS, serum T4 concentrations are within the reference of which there are several
range, while serum T3 is decreased and rT3 is increased. Serum TSH concentrations are typically variants.
normal to low (except for a transient increase that may occur during recovery). However, it is not
recommended to measure TSH in hospitalized patients unless there is a strong suspicion for thy-
roid disease. In seriously ill patients, ETS presents with low-normal T4 and significantly reduced
total T3 concentrations. Serum rT3 is increased because of slow thyroid hormone clearance and
greater than normal conversion of T4 to rT3 rather than to T3. An elevated rT3 in the appropriate
clinical setting, with appropriately suggestive laboratory test results, points to ETS syndrome.
Thyroid Tumors
Description
Masses or “nodules” in the thyroid may be associated with normal function, hyperfunction,
or hypofunction, and for that reason, they are considered apart from hyperthyroidism and
hypothyroidism. In fact, some studies suggest that 25% to 40% of the population have thyroid
nodules. Most solitary masses detected with physical examination are the dominant nodule in a
multinodular goiter, a cyst, or an asymmetric enlargement of the gland. Benign thyroid adeno-
mas account for most of the neoplastic nodules. The initial evaluation for a thyroid nodule is to
measure TSH and perform a thyroid ultrasound. If the TSH is suppressed, a nuclear scan can be
performed. Hyperfunctioning nodules appear as “hot nodules” by nuclear scan because they take
up radioactive iodine while uptake is suppressed in the remainder of the gland. A nonfunctional
(cold) nodule carries an increased risk of malignancy and should be followed with ultrasound-
guided FNA or biopsy. The morphologic variants of thyroid cancer, diagnosed with histopatho-
logic review of a biopsy specimen or aspirate (in order of frequency), are papillary carcinoma,
follicular carcinoma, medullary thyroid carcinoma, and anaplastic carcinoma.
Diagnosis
The diagnosis is established by histopathologic review of a specimen obtained with FNA or
biopsy. The accuracy of the diagnosis is increased with the use of guided ultrasound examina-
tion for sample collection. Treatment and risk of recurrence of thyroid neoplasms are assessed
by periodic measurement of tumor markers, including thyroglobulin for papillary and follicular
carcinomas, and calcitonin for MTC.
ADRENAL CORTEX
The adrenal cortex secretes many steroid hormones that have a wide variety of physiologic effects.
The hormones can be grouped into 3 major categories: glucocorticoids, mineralocorticoids, and
sex steroids that include androgens, progestogens, and estrogens. The glucocorticoids and min-
eralocorticoids are collectively known as corticosteroids. Steroid hormones are synthesized from
cholesterol in the adrenal glands and in the gonads. They are transported in the blood bound to
The adrenal cortex carrier proteins, such as albumin and hormone-binding globulins, or as free hormone. Steroids
secretes hormones that may be modified with glucuronate or sulfate to increase their water solubility and permit excretion
can be grouped into via the kidneys or the gastrointestinal tract. The percentage of steroid hormone that is bound to
3 major categories: protein varies with the hormone affinity for carrier proteins and ranges from 60% to nearly 100%.
glucocorticoids, Quantitatively, the glucocorticoids and mineralocorticoids are the most important group of hor-
mineralocorticoids, and mones produced by the adrenal cortex. The major corticosteroids are cortisol (a glucocorticoid)
sex steroids that include and aldosterone (a mineralocorticoid). The synthesis and metabolism of the steroid hormones are
androgens, progestogens, illustrated in Figure 22–4. The liver is the main site for conjugation of steroid hormones, and the
and estrogens. kidney excretes approximately 90% of the conjugated steroids. Glucocorticoids alter carbohydrate
metabolism by increasing gluconeogenesis and decreasing glucose utilization. Additional effects
include the inhibition of amino acid uptake and protein synthesis in peripheral tissues. Mineralo-
corticoids promote sodium conservation and potassium loss and thereby considerably influence
the retention or loss of fluid. Among the naturally occurring mineralocorticoids, aldosterone has
the highest mineralocorticoid activity followed by deoxycorticosterone and corticosterone.
Secretion of adrenal glucocorticoids and adrenal androgens is regulated by corticotropin
(ACTH) that is secreted by the pituitary gland (Figure 22–5). ACTH also plays a minor role
in aldosterone and mineralocorticoid production. Their synthesis is primarily controlled by a
Cortisol concentrations different pathway known as the renin–angiotensin system. The hypothalamic–pituitary–adrenal
are highest in the early (HPA) axis begins with the episodic release of corticotropin-releasing hormone (CRH) from the
morning between 4 and hypothalamus. CRH stimulates the episodic release of ACTH from the pituitary. ACTH then
8 AM and about 25% stimulates the adrenal cortex to produce cortisol in a diurnal or circadian manner. Cortisol con-
lower in the late evening. centrations are highest in the early morning between 4 and 8 am and about 25% lower in the late
Physical and mental evening. Physical and mental stress can elevate cortisol concentrations and blunt the circadian
stress can elevate cortisol rhythm. ACTH release is under negative feedback control from the cortisol fraction not bound
concentrations and blunt to proteins. Two adrenal sex steroids dehydroepiandrosterone (DHEA) and androstenedione are
the circadian rhythm. also stimulated by ACTH as well as other hormones including insulin-like growth factor-1 and
gonadal steroids. Adrenal androgen production reaches a peak in the second decade, with a rise
during late childhood. It gradually decreases and reaches a low level in the elderly.
17α-Hydroxylase 17, 20-Lyase
Cholesterol
Cholesterol
desmolase
Pregnenolone 17-OH pregnenolone Dehydroepiandrosterone
3β-Hydroxysteroid
dehydrogenase
Progesterone 17-OH progesterone Androstenedione
21-Hydroxylase
11-Deoxycortisol Sex steroids

11β-Hydroxylase 11-Deoxycorticosterone
18-Hydroxylase
Corticosterone Cortisol
18-Oxidase
Glucocorticoids
18-OH corticosterone
Aldosterone
synthase
Aldosterone
Mineralocorticoids
FIGURE 22–4 The synthesis and metabolism of steroid hormones of the adrenal cortex.
Physical and emotional stress,

hypoglycemia, and other stimuli
[+]
[–] [–]
Hypothalamus
Corticotropin-releasing
hormone (CRH)
[+]
[–]
Pituitary
Adrenocorticotropic
hormone (ACTH)
[+]
Sex steroids Cortex of adrenal gland Cortisol
FIGURE 22–5 Hypothalamic–pituitary–adrenal cortex interactions. [+] Stimulation; [−] inhibition.

Liver
Angiotensinogen
[+]
Angiotensin I
Angiotensin-
converting
enzyme (lungs)
Angiotensin II
[+]
Aldosterone Adrenal cortex Renin
[+]
Normal or
Increased circulating elevated [–]
Renal reabsorption of [+] blood volume and Kidney:
sodium and excretion blood pressure Or Juxtaglomerular
of potassium apparatus
Increased sodium
concentration Not yet normal [+]
FIGURE 22–6 The renal and adrenal interactions in the renin–angiotensin system.
As shown in Figure 22–6, aldosterone secretion is primarily controlled by the renin–angiotensin

system. Renin is an enzyme synthesized and stored in cells of the juxtaglomerular afferent arterioles
of the renal glomeruli. The circulating renin hydrolyzes angiotensinogen to produce angiotensin I,
which is rapidly converted to angiotensin II by angiotensin-converting enzyme. Angiotensin II then
stimulates the cells of the adrenal cortex to produce aldosterone. Angiotensin II is also a potent
vasoconstrictor. The primary stimuli for renin release are decreased perfusion of the kidney and a
negative sodium balance.
Laboratory Tests
The functional status of the adrenal cortex can be evaluated by measuring the circulating con-
centrations of components of the HPA axis and the renin–angiotensin system. In addition to
Because the secretion measurement of the plasma, serum, or urinary concentrations of these compounds, dynamic
of cortisol is pulsatile stimulation and suppression tests are valuable in identifying certain abnormalities.
and diurnal, a single,
random serum cortisol
Cortisol
measurement is not
usually diagnostic for Because the secretion of cortisol is diurnal and pulsatile, a single, random serum cortisol measure-
adrenal dysfunction. The ment is not useful in the diagnosis of adrenal dysfunction. However, a decreased early morning
24-hour urinary excretion serum cortisol measurement may suggest adrenal insufficiency. The 24-hour urinary excretion
of cortisol is a reliable of cortisol, an index of plasma-free cortisol during that 24-hour time frame, is a reliable gauge
gauge of excess cortisol of excess cortisol secretion by the adrenal cortex. This is because taking an average urine cortisol
secretion by the adrenal concentration over 24 hours eliminates the need to account for diurnal variation as in a single
cortex. serum collection. Urinary free cortisol (UFC) measurements should not be used in patients with
renal impairment. Late-night salivary cortisol is also a measure of free cortisol since the binding
protein does not cross into saliva. Salivary cortisol concentrations correlate well with serum con-
centrations and are not influenced by changes in cortisol-binding protein concentrations. Mea-
surement of late-night salivary cortisol is an acceptable screening method for hypercortisolism.
Low-dose Dexamethasone Suppression Tests

Dexamethasone is a synthetic glucocorticoid more potent than cortisol that, when given orally or
IV, suppresses ACTH and CRH secretion. It can be administered as 1 mg at midnight in an over-
night dexamethasone suppression test, or in a 2-day low-dose dexamethasone suppression test
(LDDST) by giving the patient 0.5 mg every 6 hours for 8 doses. If the dexamethasone is effective,
it will suppress ACTH secretion and, thereby, suppress cortisol production. Patients with Cush-
ing disease normally do not show suppression of cortisol synthesis after either dexamethasone
administration for the LDDST or dexamethasone in the overnight suppression test.
ACTH
In patients with an abnormal dexamethasone suppression test, ACTH measurements are impor-
tant to determine whether Cushing syndrome is ACTH-dependent. The optimum time of day for
determination of plasma ACTH concentration in patients with suspected Cushing syndrome is
between midnight and 2 am when the plasma-circulating concentrations of ACTH and cortisol
are at their lowest point. At this time, the ability to detect an abnormality in ACTH secretion is the
greatest. In patients with suspected adrenal insufficiency, ACTH should be measured in the morn-
ing during its peak. Suppressed morning ACTH may indicate excess adrenal cortisol secretion.
ACTH Stimulation Test

The ACTH stimulation test is the most useful test in diagnosis of adrenal insufficiency. It assesses
the secretory response of the adrenal cortex to an ACTH-like stimulus. A 250-mg dose of an
ACTH analog, Cortrosyn or cosyntropin, is administered IV or IM and serum cortisol is mea-
sured at 0, 30, and 60 minutes after injection. Normally, ACTH analogs will stimulate production
of cortisol to concentrations >20 mg/dL. Lack of rise in cortisol after ACTH stimulation may
indicate primary and secondary adrenal insufficiency.
CRH Stimulation Test

CRH can be administered intravenously to determine the response of the pituitary to stimula-
tion by hypothalamic hormone. The CRH stimulation test can be used to determine the source
of adrenal insufficiency in patients with an abnormal ACTH stimulation test. Synthetic CRH is
administered IV, and then ACTH and cortisol are measured at 0, 30, 60, 90, and 120 minutes after
injection. Normal patients will respond to CRH by stimulating ACTH and cortisol production.
Patients with primary adrenal insufficiency will have elevated ACTH, but no cortisol production,
while patients with secondary disease will have both low ACTH and cortisol.
Aldosterone
The most important test to establish a diagnosis of hyperaldosteronism or hypoaldosteronism is
plasma aldosterone. Aldosterone can be measured in the plasma as the unmodified hormone, and
in the urine as the 18-glucuronide conjugated metabolite of aldosterone. Screening for primary
aldosteronism should include the determination of the ratio of plasma aldosterone concentration
(PAC)/plasma renin activity (PRA), that is, the aldosterone to renin ratio (ARR). Aldosterone and
renin concentrations are affected by numerous conditions and medication. Therefore, it is recom-
mended that testing be performed under the following conditions: patients with normal potas-
sium concentrations, patients with normal salt intake, patients removed from drugs that affect
the ARR, and midmorning collection in patients who have been ambulatory for at least 2 hours.
Renin
PRA is assayed by measuring its ability to convert angiotensinogen to angiotensin I, which is
then quantitated by an immunoassay. When primary aldosteronism is present, often because of
either resistant or severe hypertension, the ARR will be elevated. Renin mass can also be assessed
directly by immunoassay, to determine the direct renin concentration (DRC). Although the ARR
can be calculated using DRC or PRA, fewer studies have investigated the utility of DRC. Renin
specimens should not be stored refrigerated or on ice prior to analysis as cold temperatures pro-
mote secretion of prorenin, which falsely elevates results. See the section “Aldosterone” for recom-
mended testing conditions.
Steroid Measurements to Identify Enzyme Deficiencies

in Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia (CAH) represents a spectrum of diseases resulting from enzyme
deficiencies that impair normal hormone synthesis in the adrenal cortex. The decrease in cortisol
Cushing syndrome is production in most forms of CAH leads to an overproduction and shunting into the androgen
a disorder of excess synthesis pathway. These disorders are described in the section “Alterations in the Synthesis of
cortisol production, Glucocorticoids, Mineralocorticoids, and Sex Steroids: Congenital Adrenal Hyperplasia.” The
which is more commonly assays used to identify the specific enzyme deficiencies include measurement of 17-hydroxypro-
ACTH-dependent than gesterone (17-OHP), either unstimulated or after ACTH stimulation, 17-hydroxypregnenolone
ACTH-independent. after ACTH stimulation, deoxycorticosterone, 11-deoxycortisol, and several androgens (andro-
stenedione, DHEA, and testosterone). DNA-based tests also can be used to identify certain gene
mutations that result in enzyme deficiencies found in CAH.
Hyperfunction Involving Glucocorticoids With or Without

Mineralocorticoids: Cushing Syndrome
Description
Cushing syndrome is a disorder of excess cortisol production, which is more commonly ACTH-
dependent than ACTH-independent. The early clinical features of Cushing syndrome are hyper-
tension and weight gain. Truncal obesity with a round face, often known as a “moon facies,” and
an accumulation of fat in the posterior neck and regions of the back close to the neck, known as a
“buffalo hump,” appear with progression of the disease. Decreased muscle mass and proximal limb
weakness occur from atrophy of muscle fibers induced by the high level of cortisol, which inhibits
protein synthesis and uptake of glucose by the cells. Patients with Cushing syndrome often have
elevated blood glucose levels with glucosuria. Other clinical signs and symptoms include striae of
the skin, osteoporosis, hirsutism, menstrual abnormalities in women, and mental status changes
involving mood swings with depression.
Cushing syndrome is separated into 3 main disease entities, with increased synthesis and loss
of circadian rhythm resulting in excess cortisol production by the adrenal cortex as the common
theme. Independent of the 3 naturally occurring forms of Cushing syndrome, the administration
of glucocorticoids as a medication is a common cause of Cushing syndrome. This should be evident
from the medication history. Endogenous forms of Cushing syndrome are described as follows:
• Cushing disease is the most common form of Cushing syndrome and is 4- to 6-fold more
prevalent in women. It is caused most often by small ACTH-secreting tumors in the
pituitary (<1 cm in size) known as microadenomas. These adenomas can be detected
with various radiographic techniques after appropriate hormone tests suggest a pituitary
etiology. On rare occasions, the tumors are large and present as macroadenomas.
• Adrenal Cushing syndrome—This form of Cushing syndrome is most commonly associated
with a benign or malignant tumor in the adrenal cortex. Adrenal adenomas synthesize
cortisol efficiently, but adrenal carcinomas often synthesize cortisol inefficiently and
overproduce sex steroids, resulting in virilization.
• Cushing syndrome from ectopic ACTH production—Small cell lung carcinoma and bronchial
carcinoid patients account for most of the Cushing syndrome cases in this category. This
form of Cushing syndrome is more common in men because of the higher incidence of
lung cancer in men. In the absence of signs and symptoms specifically associated with the
lung carcinoma or carcinoid, these patients are clinically indistinguishable from those with
pituitary Cushing disease. Because the syndrome often appears in patients with significant
clinical manifestations of cancer, the symptoms from ectopic ACTH production often go
unrecognized. Another very rare cause of Cushing syndrome with an ectopic focus and The strategy for the
high serum ACTH level is a tumor, most frequently a bronchial carcinoid tumor, which diagnosis of Cushing
secretes CRH instead of ACTH. syndrome, and the
subsequent identification
of 1 of the 3 forms of
Diagnosis
Cushing syndrome,
The diagnosis of Cushing syndrome, or Cushing disease, discussed below collectively as Cushing involves tests to
syndrome, requires evidence of increased cortisol production and loss of suppression of corti- confirm endogenous
sol synthesis or loss of cortisol diurnal variation. Screening for Cushing syndrome is difficult hypercortisolism and then
because 1) its prevalence is low; 2) several common conditions can produce biochemical and clin- to determine whether
ical signs of hypercortisolism in the absence of Cushing syndrome; and 3) the screening tests have the disease is ACTH-
a high rate of false-positive results for Cushing syndrome. For this reason it is recommended that dependent.
a careful history be taken to exclude exogenous causes of hypercortisolism and that only patients
with high clinical suspicion, such as those with unusual symptoms, for example, osteoporosis and
less than 40 years old, children with a decrease in height and increase in weight percentiles, severe
symptoms, or adrenal tumors visualized by imaging, be screened for Cushing syndrome.
The strategy for the diagnosis of Cushing syndrome, and the subsequent identification of 1
of the 3 forms of Cushing syndrome, involves tests to confirm endogenous hypercortisolism and
then to determine whether the disease is ACTH-dependent. ACTH concentrations are low in
patients with adrenal tumors, but normal or elevated with pituitary or ectopic ACTH-producing
tumors. Table 22–2 summarizes the laboratory evaluation for Cushing syndrome.
The following steps to diagnose and differentiate Cushing syndrome can be made using first-
line and then second-line diagnostic tests, respectively.
Diagnostic Evaluation
It is recommended that at least one of the following testing strategies be performed to diagnose
patients with Cushing syndrome:
1. The 24-hour UFC or late-night salivary cortisol is a sensitive screening test for patients
with clinical signs and symptoms of Cushing syndrome. Because of the variability
of the cortisol concentrations, the UFC or salivary cortisol should be elevated on
at least 2 separate occasions in order to proceed with the diagnostic evaluation for
Cushing syndrome. If the UFC or salivary cortisol is normal in a high-risk patient, an
endocrinologist should be consulted for further studies. A low-risk patient with normal
results should be rescreened in 6 months if signs or symptoms persist or worsen.
2. In unhealthy, but not critically ill, patients, and/or those with abnormal renal function,
or disrupted sleep patterns, physicians should use one of the LDDSTs to screen for
Cushing syndrome. If cortisol production is suppressed by a low dose of dexamethasone
in an overnight or 48-hour test, Cushing syndrome is ruled out. If the clinical suspicion
TABLE 22–2 Laboratory Evaluation for Cushing Syndrome

Laboratory Test Pituitary Cause Adrenal Cause Ectopic ACTH Secretion
24-Hour urine-free Elevated Elevated Elevated

cortisol or late-night
salivary cortisol
Low-dose No cortisol suppression No cortisol No cortisol suppression

dexamethasone suppression
suppression test
Plasma ACTH Elevated or inappropriately Low For ectopic ACTH-secreting tumors,

normal ACTH is elevated and often at
higher levels than found in Cushing
disease (pituitary cause)
Imaging study MRI: pituitary tumor may CT: adrenal A tumor outside the adrenal may be
be demonstrated tumor may be demonstrated
demonstrated
ACTH, adrenocorticotropic hormone (corticotropin).
for Cushing syndrome is still high in the presence of a nonsuggestive result for Cushing
syndrome, further evaluation by an endocrinologist may be useful. A lack of cortisol
suppression after a low dose of dexamethasone is suggestive of Cushing syndrome.
Patients taking oral estrogens should not be screened with the LDDST, as estrogens
stimulate the liver to synthesize cortisol-binding globulin (CBG) and elevate serum
cortisol concentrations, causing false-positive screening results. Further, critically ill
patients synthesize less CGB and albumin, leading to lower serum cortisol concentrations,
and possibly false-negative screening results.
3. Other clinical conditions that cause hypercortisolism should be ruled out. These include
alcoholism, severe obesity, pregnancy, depression, diabetes mellitus, and glucocorticoid
resistance.
Tests to Identify Etiology

Once Cushing syndrome is confirmed, a series of tests can then be performed to locate the cause
of the hypercortisolism.
Adrenal insufficiency 1. Plasma ACTH testing should be performed to determine the etiology of the
can be either primary, hypercortisolism. If ACTH is suppressed, an adrenal source is suspected. If ACTH is high
from destruction of or inappropriately normal, the patient may have a pituitary adenoma.
the adrenal cortex by a 2. If an ACTH-secreting pituitary adenoma is suspected, MRI of the pituitary gland to
local disease process or identify a mass or petrosal sinus blood sampling to collect samples coming directly out of
a systemic disorder, or the pituitary after CRH stimulation is used as a confirmation test. A ratio of petrosal ACTH
secondary from pituitary to serum ACTH above suggested cutoff values before and after CRH administration is
or hypothalamic disease consistent with a pituitary cause.
that reduces stimulation 3. If the ACTH is suppressed, a CT of the adrenal glands is likely to be informative to identify
of the adrenal gland.
an adrenal tumor and indicate an adrenal cause.
4. If an ectopic ACTH-secreting tumor is suspected, this can be challenging to locate, but
imaging studies are often valuable. Chest, pancreas, colon, and gall bladder carcinomas
have been shown to be sources of cortisol secretion.
“Pseudo-Cushing syndrome,” which can be produced by alcohol abuse and other disorders,
mimics both the clinical and biochemical features of the true syndrome.
Hypofunction Involving Glucocorticoids With or Without

Mineralocorticoids: Adrenal Insufficiency
Description
Adrenal insufficiency can be either primary, from destruction of the adrenal cortex by a local
disease process or a systemic disorder, or secondary from pituitary or hypothalamic disease that
reduces stimulation of the adrenal gland. The most common causes of primary adrenal insuf-
ficiency, in which all classes of adrenal cortical steroids are deficient, are autoimmune adrenalitis
and tuberculosis in endemic regions. In this particular disorder, the adrenal medulla and its cat-
echolamine synthesis are spared. In other primary adrenal insufficiency disorders in which the
adrenal gland is damaged, the adrenal medulla may be damaged along with the adrenal cortex.
In secondary adrenal insufficiency, in which there is deficient stimulation of the adrenal gland
because of pituitary or hypothalamic abnormalities, the adrenal medulla is not affected and aldo-
sterone deficiency is not usually present. Aldosterone secretion is more dependent on angio-
tensin II stimulation of the adrenal cortex than on stimulation of the adrenal cortex by ACTH
(as discussed below).
• Primary adrenal insufficiency—There are many causes of primary adrenal insufficiency.
Dysfunction in 1 or more sites in the HPA axis is the major cause of the primary adrenal
insufficiency. Chronic primary adrenal insufficiency, also known as Addison disease, occurs
mostly in adults. The most common causes are autoimmune disease (Western world) and
tuberculous adrenalitis (worldwide). Other causes of primary adrenal insufficiency include
fungal or viral infections (ie, histoplasmosis or HIV), and anatomic destruction of the
adrenal glands through surgery, hemorrhage, or metastatic carcinomas. Primary adrenal
insufficiency is characterized by hyperpigmentation of the skin and mucous membranes.

The lack of negative feedback from adrenal cortisol leads to increased ACTH production by
the pituitary. A degradation product of ACTH and its prohormone pro-opiomelanocortin is
melanocyte-stimulating hormone (MSH). MSH stimulates melanin production and induces
melanocyte hyperpigmentation. Hyperkalemia and hypotension may be present if there
is deficient aldosterone (see the sections “Primary Hypoaldosteronism” and “Secondary
Hypoaldosteronism”). Primary adrenal insufficiency usually has a gradual onset but may
occur abruptly with a stressor such as critical illness or surgery. The rapid onset form of
adrenal insufficiency is often the result of adrenal hemorrhage or thrombosis that impairs
blood supply to the gland.
Acute primary adrenocortical failure (also called Addisonian crisis) can be triggered by
a severe infection, sepsis, or abrupt withdrawal of steroids. It is a life-threatening emergency,
characterized by abnormal electrolytes (critically high potassium and low sodium),
hypotension, and hypoglycemia. Sudden death can occur if it is not treated promptly.
• Secondary adrenal insufficiency—A deficiency of ACTH secretion from any cause can
lead to adrenal insufficiency. Long-term glucocorticoid therapy can result in prolonged
suppression of CRH from the hypothalamus and ACTH from the pituitary and transient Adrenal insufficiency
adrenal insufficiency. Hypopituitarism as a result of postpartum hemorrhage (Sheehan may involve a deficiency
syndrome), radiation, surgery, or injury may result in decreased ACTH production leading of glucocorticoids or
to reduced glucocorticoid synthesis. both glucocorticoids
and mineralocorticoids.
Depending on the extent
Diagnosis
of adrenal hormone
The management of adrenal insufficiency first requires determination of the disease source (ie, deficiency, patients
primary or secondary), followed by an identification of the specific cause for the adrenal insuf- may have decreased
ficiency. The tests that are useful in the diagnosis of primary and secondary adrenal insufficiency serum cortisol alone
are shown in Table 22–3. Adrenal insufficiency may involve a deficiency of glucocorticoids or or in combination with
both glucocorticoids and mineralocorticoids. Depending on the extent of adrenal cortex dam- decreased plasma
age, patients may have decreased serum cortisol and/or decreased plasma aldosterone levels. aldosterone.
TABLE 22–3 Laboratory Evaluation for Adrenal Insufficiency

Laboratory Test Primary Adrenal Insufficiency Secondary Adrenal Insufficiency
ACTH stimulation test Synthetic ACTH does not stimulate cortisol If the secondary adrenal insufficiency is mild or of recent onset,
secretion because the dysfunctional adrenal there is an increase in cortisol secretion; in chronic secondary
cortex is already maximally stimulated by adrenal insufficiency plasma cortisol is minimally increased
endogenous ACTH after administration of ACTH because the adrenal cortex is
atrophied from a long-term lack of stimulation by ACTH
Serum cortisol measured Low Low
between 8 and 9 AM
Plasma ACTH Elevated, because feedback inhibition by Low, because the origin of the disorder is in the
adrenal cortisol is absent hypothalamus or pituitary
Plasma aldosterone Low in cases in which injury to the adrenal gland Often normal, although it may be depressed if there is
impacts both cortisol and aldosterone production significant atrophy of the adrenal glands as a result of chronic
lack of stimulation by ACTH
CRH stimulation test Not necessary This test can distinguish between ACTH deficiency (from the
pituitary) and deficiency of CRH (from the hypothalamus);
plasma ACTH and cortisol are measured after administration
of CRH; if secondary adrenal insufficiency is the result of a
hypothalamic disorder, the CRH will produce an increase in
plasma ACTH and cortisol
Adrenal autoantibody tests Serum tests that detect titers of adrenal Not necessary
autoantibodies are available for the confirmation
of autoimmune-mediated primary adrenal
insufficiency. The most commonly ordered is the
21-hydroxylase antibody test
ACTH, adrenocorticotropic hormone (corticotropin); CRH, corticotropin-releasing hormone.
The ACTH stimulation test is the most specific test to confirm a diagnosis of adrenal insuffi-
ciency. Patients with primary adrenal insufficiency do not usually show cortisol secretion fol-
lowing ACTH stimulation because the defect is within the adrenal gland. Patients with mild or
recent onset of secondary adrenal insufficiency (and a still viable adrenal cortex) respond to the
ACTH because the defect is not within the adrenal gland. A chronic lack of stimulation of the
adrenal cortex by ACTH in secondary adrenal insufficiency can result in cortical atrophy and
limited cortisol production following ACTH stimulation. The CRH stimulation test can distin-
guish between secondary adrenal insufficiency caused by ACTH deficiency and that caused by
CRH deficiency. Plasma ACTH and serum cortisol are measured after administration of CRH;
increases are observed in hypothalamic disorders, but not in pituitary disorders. A serologic test
for antiadrenal antibodies is useful to determine if autoimmune adrenalitis is the cause of primary
adrenal insufficiency.
Hyperfunction and Hypofunction Involving Mineralocorticoids:

Hyperaldosteronism and Hypoaldosteronism
Aldosterone is a mineralocorticoid produced in the adrenal glands. It is largely responsible for
regulating sodium retention and water resorption, and thereby control of blood volume. It also
Aldosterone is a
promotes the excretion of potassium into the urine. Aldosterone concentration in the blood is
mineralocorticoid
produced in the adrenal
regulated by the renin–angiotensin aldosterone (RAA) system (Figure 22–6). In response to
glands. It is largely decreased blood volume, the juxtaglomerular apparatus of the kidney secretes renin, which con-
responsible for sodium verts angiotensinogen to angiotensin I. Angiotensin I is converted to angiotensin II by angioten-
retention and water sin-converting enzyme in the lungs. Angiotensin II is a potent vasoconstrictor and also stimulates
resorption, and thereby the adrenal glands to secrete aldosterone, which then acts to increase blood volume by promoting
control of blood volume. sodium retention in exchange for potassium that is lost into the urine.
It also promotes the Aldosterone concentrations in disease may be high (hyperaldosteronism) or low (hypoal-
excretion of potassium dosteronism). An abnormal (high or low) PAC may be the result of a defect originating inside
into the urine. (primary disorder) or outside (secondary disorder) of the adrenal gland (Table 22–4).
TABLE 22–4 Laboratory Evaluation for Hyperaldosteronism and Hypoaldosteronism

Secondary (Renin-mediated) Primary Secondary
Laboratory Test Primary Hyperaldosteronism Hyperaldosteronism Hypoaldosteronism Hypoaldosteronism
Serum potassium Usually low, but a low- Usually low, but a low- Usually elevated Usually elevated
sodium diet may result sodium diet may result
in a normal value in a normal value
Serum sodium Normal Normal or mildly elevated Usually low Low or low normal
Plasma aldosterone Elevated in midmorning Elevated in midmorning Usually low in Usually low in the absence
samples collected from samples collected from the absence of of medications that affect
normokalemic patients normokalemic patients medications that activity
recumbent 2 h on an recumbent 2 h on an affect activity
unrestricted sodium diet unrestricted sodium diet
(>100 mmol/day) for at least (>100 mmol/day) for at least
3 days, and in the absence of 3 days, and in the absence of
inhibiting medications inhibiting medications
Plasma renin activity Low for most causes of Elevated when there is Normal or elevated Hypoaldosteronism may
or direct renin hyperaldosteronism in decreased perfusion of in the absence of be secondary to a variety
normokalemic patients with the kidneys, a common medications that of disorders associated
normal renal function in the cause of secondary affect activity with low renin production,
absence of medications that hyperaldosteronism in the and in these disorders
affect activity absence of medications that the renin is low; with
affect activity other causes of secondary
hypoaldosteronism, the renin
may be normal or elevated
PAC/PRA (DRC) ratio Elevated (see appropriate Normal to low N/A N/A
conditions for each test above)
Primary Hyperaldosteronism
In this disorder, there is excess secretion of aldosterone as a result of an abnormality within the
adrenal gland. Most often, hyperaldosteronism is caused by bilateral hyperplasia of the adrenal
glands or by an aldosterone-secreting adrenal adenoma, resulting in a disorder known as Conn
syndrome. Less often, primary hyperaldosteronism is a result of primary (unilateral) adrenal
hyperplasia or a cancerous tumor. In primary hyperaldosteronism, the PRA is low because aldo-
sterone stimulates high sodium concentrations and increased blood volume triggers downregu-
lation of renin secretion. The ratio of the PAC/PRA is widely used as a screening test for primary
aldosteronism in hypertensive patients. A ratio of greater than 30 when PAC is measured in con-
ventional units (ng/dL) and 750 when PAC is in SI units (pmol/L) is the most commonly used
cutoff to identify patients with this disorder. Because of the effect of numerous drugs, diet, and
comorbidities on PAC and PRA concentrations, it is recommended that along with an elevated
ratio, patients also have PAC concentrations above 15 ng/dL. Some laboratories use DRC as
an indirect measure of renin activity. A few studies have published recommended diagnostic
cutoffs for the PAC/DRC ratios for primary hyperaldosteronism, but they have not yet been
universally accepted.
Secondary Hyperaldosteronism
In this disorder, there is excess secretion of aldosterone as a result of an abnormality outside the
adrenal gland. It is much more common than primary hyperaldosteronism. Decreased renal per-
fusion is the most common cause of secondary hyperaldosteronism. The decreased blood flow
into the kidney results in an elevation of the PRA. The elevation in plasma renin level (as shown in The clinical and
Figure 22–6) produces the increase in aldosterone. Congestive heart failure, nephrotic syndrome, laboratory features
cirrhosis of the liver, and other hypoproteinemic conditions in which there is chronic depletion common to both
of plasma volume can produce an elevation in plasma aldosterone. primary and secondary
The clinical and laboratory features common to both primary and secondary hyperaldo- hyperaldosteronism
steronism usually include hypertension associated with hypervolemia and low or low-normal include hypertension
concentrations of serum potassium. The serum sodium also may be slightly elevated. Additional associated with
clinical features include nocturnal polyuria, polydipsia, and weakness from the low potassium hypervolemia and
concentrations. low or low-normal
concentrations of
Primary Hypoaldosteronism serum potassium.
This disorder is much less common than primary hyperaldosteronism. Primary hypoaldosteron-
ism is most often a result of destruction of the adrenal gland from various causes (as noted in the
section “Hypofunction Involving Glucocorticoids With or Without Mineralocorticoids: Adrenal
Insufficiency”), including autoimmune adrenalitis, adrenal infection by tuberculosis, metastatic
tumors to the adrenal, adrenalectomy, CAH associated with low aldosterone production (see the
section “Alterations in the Synthesis of Glucocorticoids, Mineralocorticoids, and Sex Steroids:
Congenital Adrenal Hyperplasia”), and hemorrhage into the adrenal gland. There is an additional
disorder associated with primary hypoaldosteronism, known as pseudohypoaldosteronism, in
which the tissues are resistant to the action of aldosterone. Low blood volume and/or sodium due
to lack of aldosterone action upregulates renin synthesis, which subsequently upregulates aldoste-
rone. Therefore, these patients have significantly elevated PAC and PRA.
Secondary Hypoaldosteronism
In this disorder, aldosterone hyposecretion results from factors originating outside the adrenal
gland. One cause is a deficiency of ACTH production in the pituitary, often accompanied by defi-
ciencies of other pituitary hormones. As noted earlier, the adrenal cortex can become atrophied
as a result of a chronic lack of stimulation by ACTH, decreasing aldosterone as well as cortisol
production. Another cause is long-term glucocorticoid administration. Long-term glucocorti-
coid-induced ACTH suppression leads to adrenal atrophy and reduced aldosterone synthesis.
Secondary hypoaldosteronism can also occur as a result of deficient renin production due to renal
damage or drugs and from inhibition of angiotensin-converting enzyme by drugs. Clinical and
laboratory features common to primary and secondary hypoaldosteronism include hypotension,
which may be orthostatic, and high serum potassium levels. Slightly low serum sodium values
also may be present. The clinical signs and symptoms vary significantly and depend on the spe-
cific defect leading to the hypoaldosteronism.
Alterations in the Synthesis of Glucocorticoids, Mineralocorticoids,

and Sex Steroids: Congenital Adrenal Hyperplasia
CAH is caused by any 1 of a group of enzyme deficiencies in the biosynthetic pathways for cortisol
and aldosterone. Because cortisol production is decreased, and cortisol provides the inhibitory
feedback to the pituitary for ACTH secretion, there is an increase in ACTH and excess stimula-
tion of the adrenal glands (see Figures 22–5 and 22–6 for the regulation of cortisol and aldoste-
rone production). This results in greater flux through pathways around an existing enzymatic
defect, producing elevations in adrenal hormones whose synthesis is not affected by the enzyme
deficiency. Most of the known enzyme deficiencies in the synthetic pathways for aldosterone and
cortisol result in an elevation in sex steroid synthesis, which has a virilizing effect on the patient.
Most of the known The most common of the enzymatic defects is a deficiency of 21-hydroxylase. This deficiency
enzyme deficiencies in accounts for 90% to 95% of the cases of CAH. The clinical manifestations for 4 of the enzyme
the synthetic pathways deficiencies producing CAH are noted below. Figure 22–4 shows the intermediate compounds in
for aldosterone and the synthesis of aldosterone, cortisol, and androgens in the adrenal gland and the enzymes in the
cortisol result in an pathway, some of which may be deficient. Table 22–5 presents the laboratory evaluation for CAH.
elevation in sex steroid
synthesis, which has • 21-Hydroxylase deficiency—Deficiency of this enzyme is the most common form of CAH.
a virilizing effect on In female infants, a 21-hydroxylase deficiency usually results in hypertrophy of the clitoris
the patient. The most and pseudohermaphroditism as a result of disruption in the synthesis pathways for cortisol
common of the enzymatic and aldosterone and shunting of intermediates down the androgen synthesis pathway.
defects is a deficiency of In postpubertal females, it results in amenorrhea, infertility, and hirsutism. In males, the
21-hydroxylase. virilization results in enlargement of the external genitalia and precocious puberty. As
seen in Figure 22–4, 21-hydroxylase deficiency will result in a reduced or no synthesis of
both cortisol and aldosterone and accumulation of the 17-OHP intermediate. As a result,
if infants with this deficiency are not treated with corticosteroids, they can quickly develop
life-threatening hyperkalemia, hyponatremia, and hypotension. Because of the severe
clinical manifestations, it is recommended that all newborns be screened for 21-hydroxylase
deficiency by measurement of 17-OHP. Positive newborn screening results should be
followed with sensitive and specific confirmatory testing.
TABLE 22–5 Laboratory Evaluation for Congenital Adrenal Hyperplasia

Relevant Laboratory Findings (Focusing on Compounds Most Likely
Enzyme Deficiency to Be Measured in an Evaluation)
21-Hydroxylase Elevated: 17-hydroxyprogesterone, androstenedione, DHEA, and its sulfated

metabolite (DHEA-S) testosterone; ACTH and plasma renin activity because of
the deficiencies of cortisol and aldosterone
Decreased: aldosterone, cortisol
11-Beta-hydroxylase Elevated: 11-deoxycortisol, 11-deoxycorticosterone; 17-hydroxyprogesterone,

androstenedione, DHEA, DHEA-S, testosterone
17-Alpha-hydroxylase Elevated: aldosterone; deoxycorticosterone
Decreased: androgens, cortisol
3-Beta-hydroxysteroid Elevated: DHEA; ACTH and plasma renin activity because of the deficiencies of cortisol
dehydrogenase and aldosterone
Assays for 17-hydroxypregnenolone and 17-hydroxyprogesterone, as well as assays for

DHEA and androstenedione, are helpful in the differentiation of 3-beta-hydroxysteroid
dehydrogenase deficiency from 21-hydroxylase deficiency and 11-beta-hydroxylase
deficiency; the 17-hydroxypregnenolone to 17-hydroxyprogesterone ratio and the
DHEA to androstenedione ratio in 3-beta-hydroxysteroid dehydrogenase deficiency
are extremely high
ACTH, adrenocorticotropic hormone (corticotropin); DHEA, dehydroepiandrosterone.
• 11-Beta-hydroxylase deficiency—This is the second most common enzyme deficiency

responsible for CAH. In infancy, the clinical and laboratory features of patients with this
abnormality are largely similar to those found in patients with 21-hydroxylase deficiency.
However, deficiency of this enzyme causes accumulation of 11-deoxycorticosterone,
a potent mineralocorticoid. Thus, these patients develop mineralocorticoid-induced
hypertension and hypokalemia.
• 17-Alpha-hydroxylase deficiency—This deficiency is rare and accounts for approximately
1% of all CAH cases. In this deficiency, there is no inhibition of aldosterone synthesis,
but there is a block in the synthesis of both cortisol and sex steroids. The elevation of
aldosterone results in hyperaldosteronism that produces hypertension and hypokalemia.
In females, the androgen deficiency results in a lack of development of secondary sex
characteristics because the androgens are biochemical precursors of estrogens. In males,
pseudohermaphroditism appears.
• 3-Beta-hydroxysteroid dehydrogenase deficiency—This is another rare CAH disorder. This
enzymatic deficiency results in a metabolic block in the production of aldosterone and
cortisol, with no inhibition of the synthesis of DHEA and other androgens. In its severe
form, this enzyme deficiency manifests as early masculinization in males and amenorrhea
and pseudohermaphroditism in females, as well as life-threatening hyperkalemia,
hyponatremia, and hypotension.
ADRENAL MEDULLA
The main sites of production of the catecholamines are the brain, the chromaffin cells of the adre-
nal medulla, and the sympathetic neurons. The catecholamines include dopamine, epinephrine,
and norepinephrine as the most potent of the endogenously produced compounds. Of these, in
the adrenal medulla, epinephrine production is quantitatively the greatest. The catecholamines
have a wide variety of biological effects. They have a marked impact on the vascular system, and The catecholamines
are important in blood pressure regulation. Epinephrine influences many metabolic pathways, include dopamine,
especially carbohydrate metabolism. In some tissues, epinephrine and norepinephrine produce epinephrine, and
opposite effects. Alpha-adrenergic receptors on cells interact effectively with norepinephrine and norepinephrine as the
moderately with epinephrine, while beta-adrenergic receptors respond primarily to epinephrine most potent of the
and norepinephrine. endogenously produced
Catecholamine synthesis and metabolism in the adrenal medulla is illustrated in compounds. Of these,
Figure 22–7. The pathway begins when the amino acid tyrosine is metabolized to a catechol- in the adrenal medulla,
epinephrine production
amine, dihydroxyphenylalanine (DOPA). DOPA is then converted to dopamine, which is trans-
is quantitatively the
formed to norepinephrine, which is subsequently converted to epinephrine. Because of their
greatest.
great potency, the catecholamines must be rapidly inactivated through reuptake into storage
granules, conversion to metabolites, or excretion. Unlike the steroid hormones, catecholamines
are not bound to proteins as they circulate. In plasma, they have a very short half-life of approxi-
mately 2 minutes. Urine catecholamines, on the other hand, represent a pool of catecholamines
delivered into urine in the preceding hours. There are a number of degradative products of epi-
nephrine and norepinephrine. The compounds noted in Figure 22–7—metanephrine, normeta-
nephrine, and vanillylmandelic acid—are the ones that are measured in clinical assays to assess There are a number of
catecholamine production and degradation. degradative products
of epinephrine and
norepinephrine.
Laboratory Tests Metanephrine,
normetanephrine,
Epinephrine and Norepinephrine and vanillylmandelic
Total or fractionated (epinephrine or norepinephrine) catecholamines can be measured in plasma acid are the ones
or 24-hour urine samples. The plasma concentration reflects the rate of synthesis and release of that are measured in
catecholamines by the adrenal medulla and their half-life in the circulation. Catecholamines are clinical assays to assess
secreted into the urine as free hormones. Urinary catecholamines are extremely unstable and catecholamine production
should be acidified during or right after collection. and degradation.
Tyrosine The amino acid precursor

of the catecholamines
A catecholamine precursor of
Dihydroxyphenylalanine (DOPA) dopamine, norepinephrine,
and epinephrine
Dopamine
Norepinephrine (noradrenaline) Catecholamines with a marked

influence on metabolism, the
vascular system, and many
other sites, primarily via different
Epinephrine (adrenaline) types of adrenergic receptors
Products of metabolism resulting

Metanephrine Normetanephrine from inactivation of norepinephrine
and epinephrine
Vanillylmandelic acid (VMA)
FIGURE 22–7 Catecholamine synthesis and metabolism in the adrenal medulla.
Metanephrines (Metanephrine and Normetanephrine)

The preferred screening test for adrenal medullary neuroendocrine tumors is detection of free
metanephrines and normetanephrines in plasma. Both metanephrine and normetanephrine
undergo conjugation with sulfate or glucuronide. The metanephrines can also be measured in a
24-hour urine specimen. Urine measurements are helpful in cases where plasma metanephrines
are marginally elevated. Metanephrines are also unstable in urine and should be acidified during
or right after collection.
Vanillylmandelic Acid
This compound is the major metabolite of both metanephrine and normetanephrine. It is mea-
sured in the urine and, although it is indicative of catecholamine synthesis and metabolism, it is
inferior to urinary metanephrine quantitation for this purpose.
Pheochromocytoma
Description
A pheochromocytoma, which may be benign or malignant, is a chromaffin cell tumor of the
adrenal medulla or autonomic nervous system that secretes catecholamines. On this basis, it is
a cause of hypertension. However, it is a rare cause of hypertension with approximately 5 pheo-
chromocytomas per 100,000 hypertensive cases. Other catecholamine-secreting chromaffin cell
tumors are paragangliomas and neuroblastomas. It is essential that a pheochromocytoma be
rapidly and accurately identified in patients with hypertension because surgical resection of the
tumor, with elimination of the hypertension and its complications, is successful in at least 90%
of cases, and the disease may be otherwise fatal. The diagnosis is made most often in patients
between the ages of 30 and 60 years. The clinical features of a patient with pheochromocytoma
include, most importantly, the presence of sustained or paroxysmal hypertension. The attacks
of hypertension occur abruptly and subside slowly, with a total duration of less than 1 hour in
approximately 80% of patients. They may be precipitated by palpation of the tumor, postural It is essential that a
changes, exertion, anxiety, trauma, pain, intake of foods or beverages containing tyramine (such pheochromocytoma be
as certain cheeses, beer, and wine), and the ingestion of certain medications. Headaches are rapidly and accurately
common in patients with pheochromocytoma, and they are usually severe. Generalized sweat- identified in patients with
ing and palpitations with tachycardia occur frequently. Other common signs and symptoms are hypertension because
anxiety, chest pain, nausea, fatigue, and weight loss. Of all pheochromocytomas, approximately surgical resection of the
25% to 30% are familial and coexist with a form of multiple endocrine neoplasia (MEN), von tumor, with elimination
Hippel Lindau, neurofibromatosis, or succinate dehydrogenase (SDH) mutation (see later sec- of the hypertension
and its complications, is
tion); 10% of inherited pheochromocytomas are malignant; 10% are extra-adrenal in location
successful in at least 90%
and are called paragangliomas (and therefore 90% are in the adrenal); and 10% are bilateral and
of cases, and the disease
most of these are patients with MEN 2A.
may be otherwise fatal.
Diagnosis
As noted before, the most biologically significant catecholamines synthesized by a pheochromocy-
toma are epinephrine and norepinephrine. These compounds are metabolized into metanephrine
and normetanephrine, respectively (Figure 22–7), and both of these compounds can be metab-
olized to vanillylmandelic acid. Measurement of fractionated, free plasma metanephrines is the
preferred screening test to rule out pheochromocytoma. This test measures plasma levels of free
metanephrine and free normetanephrine as separate compounds. All patients with clinical symp-
toms and elevated plasma free metanephrines should undergo localization studies with an adrenal
CT scan or MIBG (an imaging study involving the use of the radioisotope MIBG). Follow-up test-
ing for patients with suspected pheochromocytoma and a borderline positive plasma metaneph-
rine screening test include measurement of metanephrines in a 24-hour urine sample and then
fractionated catecholamines in plasma or urine. The diagnosis of pheochromocytoma is based
on the detection of increased concentrations of urinary or plasma metanephrines, and possibly
plasma or urinary catecholamines, in the appropriate clinical setting of sustained or paroxysmal
hypertension. Adrenal CT or other radiographic techniques can be used to localize a pheochromo-
cytoma. Laboratory tests used for diagnosis of pheochromocytoma are described in Table 22–6.
TABLE 22–6 Laboratory Evaluation for Pheochromocytoma

Plasma metanephrines Measurement of fractionated, free plasma metanephrines is the preferred screening test to rule out
pheochromocytoma; low or normal metanephrine and normetanephrine concentrations reliably exclude
the diagnosis of pheochromocytoma. Several medications, including caffeine, and cigarette smoking may
interfere with the plasma metanephrine assay
Urinary metanephrines More than 95% of patients with pheochromocytoma will have an elevated concentration of metanephrine and
normetanephrine in a 24-h urine collection; the measurement of total metanephrines per gram of creatinine
can be made in a 24-h urine collection; elevated urinary metanephrines in the presence of clinical signs and
symptoms consistent with pheochromocytoma can establish the diagnosis
Plasma catecholamines Concentrations of plasma catecholamines are elevated in pheochromocytoma; if hypertension is paroxysmal
(epinephrine and norepinephrine) rather than sustained, blood must be obtained for catecholamine
measurement during a spontaneous or provoked hypertensive episode to demonstrate an elevated
plasma catecholamine level; because plasma catecholamines increase with stress, the sample for plasma
catecholamine measurement must be collected with careful regard to minimize stress to the patient; blood
is optimally obtained after at least 20 min of rest and drawn through a previously inserted venous cannula;
the medications that the patient is ingesting at the time of or immediately prior to the test may also influence
catecholamine concentrations
Urinary catecholamines Urinary catecholamines measured in a 24-h urine collection may be used in the initial assessment of suspected
pheochromocytoma; however, the test for urinary metanephrines has a higher sensitivity for detection of
pheochromocytoma
Urinary vanillylmandelic acid Urinary VMA is elevated in the majority of patients who have a pheochromocytoma, but it is less sensitive than
the test for urinary metanephrines for diagnosis of the disorder; the urinary VMA level is not needed to establish
the diagnosis; ingestion of tricyclic antidepressants and selected other medications may produce spurious results
in this assay
PARATHYROID GLANDS
This section is focused on parathyroid hormone (PTH) and disorders associated with high or
low concentrations of this hormone. Most parathyroid disorders alter calcium metabolism, and
thereby have an effect on bone. However, there are also a number of disorders associated with
hypercalcemia or hypocalcemia, or alterations in bone density, in which a change in the PTH level
is not a major factor. Therefore, in addition to hyperparathyroidism and hypoparathyroidism, this
chapter also briefly describes a few selected disorders associated with hypercalcemia, hypocalce-
mia, or altered bone density in which PTH does not play a major role.

PTH is a polypeptide secreted from the parathyroid glands. The primary function of PTH is the
regulation of the concentration of ionized calcium in extracellular fluids. An increase in secretion
of PTH produces a rise in serum ionized calcium and a decrease in the serum phosphorus con-
centration. A normal or an elevated blood calcium provides negative feedback to the parathyroid
gland to reduce the secretion of PTH (see Figure 22–8).
The resorption of bone induced by PTH is mediated by increased activity of osteoclasts. PTH
can also promote an increase in the renal tubular reabsorption of calcium.
Vitamin D is an intermediary in the action of PTH to elevate the serum calcium level. It is a
An increase in secretion fat-soluble hormone required for calcium absorption in the gut, bone metabolism, and develop-
of PTH produces a ment of cells in the immune system. Vitamin D also influences phosphorus metabolism. Vitamin
rise in serum ionized D2 is known as ergocalciferol, and vitamin D3 is known as cholecalciferol.
calcium and a decrease Food can be fortified with either vitamin D2 or D3, both of which can be used as vitamin D
in the serum phosphorus supplements. Cholecalciferol is ingested in the diet, and it is also synthesized in the skin upon ultra-
concentration. Calcitonin violet irradiation of 7-dehydrocholesterol. The cholecalciferol is transported to the liver where it is
has an opposing action hydroxylated to produce 25-hydroxycholecalciferol (25-(OH)D3). The 25-(OH)D3 has limited bio-
to PTH, but in humans logical activity, but in the kidney it undergoes further hydroxylation to form dihydroxy metabolites,
it appears to play a
the most potent of which in calcium metabolism is 1,25-(OH)2D3. An increase in this vitamin D
minor role in calcium
metabolite results in increased intestinal absorption of calcium, mobilization of calcium and phos-
homeostasis.
phorous from the bone, and increased calcium reabsorption in the kidney, all acting to elevate
plasma calcium concentrations. The production of this dihydroxy metabolite of vitamin D is regu-
lated by the need for calcium in the circulation. Decreased blood calcium results in a stimulation
of the parathyroid glands to secrete PTH that leads to the increased production of 1,25-(OH)2D3 in
the renal proximal tubules. Thus, PTH is responsible for maintaining the necessary levels of calcium
in the body by extracting sufficient calcium from the diet, resorbing it from bone, or preventing its
excretion through the renal tubules. Figure 22–8 shows the regulation of PTH secretion. Ingested
vitamin D2 is hydroxylated into 25-(OH)D2 and follows the same metabolism to 1,25-(OH)2D2.
Calcitonin has an opposing action to PTH, but in humans it appears to play a minor role in
calcium homeostasis. As a drug, its pharmacologic action is more definitive. It inhibits osteoclas-
tic bone resorption. It also decreases renal tubular reabsorption of calcium, and by these mecha-
nisms opposes the action of PTH. Calcitonin synthesis occurs in the parafollicular C cells of the
thyroid gland.
Approximately 98% of calcium is present in the body within the bones in the form of hydroxy-
apatite, a crystal lattice composed of calcium, phosphorus, and hydroxide. Of the calcium not
Only about 1% of the within the bones, about half is present in extracellular fluid and the remainder is present in a vari-
calcium in the bones is ety of tissues, particularly skeletal muscle. Only about 1% of the calcium in the bones is exchange-
exchangeable with the able with the extracellular fluid, and it is this pool that is most significantly affected by changes
extracellular fluid, and it in PTH concentration. Calcium exists in the plasma in 3 distinct forms: free or ionized calcium,
is this pool that is most protein-bound, and complexed with anions. Ionized calcium is the physiologically active form of
significantly affected by calcium and accounts for approximately 45% to 50% of the total calcium in the plasma. Another
the level of PTH. 40% to 45% of calcium in the plasma is bound to plasma proteins. The protein that binds most of
this calcium is albumin, but calcium also binds to some globulins. The remaining 5% to 15% of
the total calcium forms a complex with a variety of anions. The most commonly found complexes
are calcium phosphate and calcium citrate. The distribution of the 3 forms of calcium changes
with alterations in pH in the extracellular fluid and with changes in plasma protein concentration.
Low plasma calcium
[+]
[+]
Parathyroid glands
[–]
Parathyroid hormone (PTH)
[+] [+] [+]
Increased production Increased renal Increased calcium

of 1,25-dihydroxyvitamin D tubular reabsorption resorption from bone
by the kidneys of calcium
Increased intestinal Normal or elevated

absorption of calcium plasma calcium
Or
Not yet normal

plasma calcium
FIGURE 22–8 The regulation of parathyroid hormone secretion. [+] Stimulation; [−] inhibition.
In general, the serum ionized calcium increases in acidosis and decreases in alkalosis because
calcium more easily binds proteins under alkalotic conditions. An increase in the concentration
of plasma proteins that bind calcium results in a corresponding increase in total calcium, and a
decrease in the plasma proteins may result in a decrease in total calcium.
The metabolism of phosphorus is linked to the metabolism of calcium. About 85% of the
phosphorus in an adult is present in the bone as part of hydroxyapatite. Most of the remaining
phosphorus in the body is within phospholipids, proteins, carbohydrates, nucleotides, and other
important biochemical compounds. Phosphorus is present in virtually all foods, and dietary defi-
ciencies do not occur. The phosphorus in the extracellular fluid exists primarily as HPO42− and
H2PO4−, which are collectively known as inorganic phosphorus. The relative amounts of these 2
phosphate anions are pH-dependent. Food ingestion can alter the serum inorganic phosphorus
concentration significantly, with an increase in serum phosphorus concentration following the
ingestion of phosphate-rich food. Because of the rapidly growing skeletal system, phosphorous
demands and serum concentrations are significantly higher in children.
Laboratory Tests
Calcium
Whole blood, serum, or plasma specimens can be used for measurement of total calcium levels.
For accurate measurement of the ionized or free form of calcium, the specimen must be trans-
ported on ice and must not be exposed to air.
Inorganic Phosphorus
About 15% of the inorganic phosphorus, predominantly HPO42− and H2PO4−, in the plasma is
protein-bound, and the remainder is free or complexed to another ion. Organic phosphorus
(not measured in the assay for inorganic phosphorus) refers to the phosphorus within phospho-
lipids, proteins, carbohydrates, nucleic acids, and other organic substances.
PTH
The most important test in the differential diagnosis of hypercalcemia is the assay for serum PTH.
The biological activity of PTH resides in the first 34-amino terminal amino acids.
The intact hormone (iPTH) with 84 amino acids accounts for much of the circulating
PTH, but there are many circulating PTH fragments. The assay for iPTH has largely super-
seded earlier tests that recognize numerous inactive circulating PTH fragments. One fragment
of interest is the fragment of PTH representing amino acids 7-84 that is present in high con-
centrations during renal disease and capable of antagonizing the PTH receptor. Many newer
iPTH assays still recognize the 7-84 fragment, along with the intact molecule, and therefore
may not be less clinically informative. An assay for whole PTH is available that only recognizes
the 1-84 amino acid PTH.
Intraoperative PTH Assay

Primary hyperparathyroidism requiring parathyroidectomy is a challenge because of variability
in the location and number of parathyroid glands. Of parathyroid adenomas, 15% to 20% are
ectopic, and not adjacent to the thyroid gland, and approximately 5% of patients have 5 para-
thyroid glands rather than 4. The success of parathyroid surgery has been improved by intra-
operative PTH measurement. The relatively short half-life of PTH has allowed for surgeons to
The success of measure plasma PTH concentrations before and after excision of parathyroid tumors in surgery.
parathyroid surgery A decrease in PTH of >50% after resection suggests complete resection of the tumor. Use of this
has been improved intraoperative test has resulted in a higher incidence of complete removal of hypersecreting para-
by intraoperative PTH thyroid gland tissue, reduced the need for extensive exploration of the neck, and decreased the
measurement. The need for repeat surgery.
intraoperative PTH
assay is used to detect
decreases in plasma PTH
Vitamin D
levels following excision The quantitation of selected vitamin D metabolites is useful in assessing vitamin D metabolism.
of parathyroid tumors in Vitamin D metabolites of greatest relevance to calcium metabolism include 25-(OH)D3 (also
surgery. known as 25-hydroxyvitamin D) and 1,25-(OH)2D3 (also known as 1,25-dihydroxyvitamin D).
Currently the most commonly ordered test, as a screening test for vitamin D deficiency, is the
total 25-(OH) vitamin D, which is the sum of the 25-(OH) vitamin D2 and 25-(OH) vitamin D3
concentrations in serum. Recently, tandem mass spectrometry has increased in use and allowed
for measurement of vitamin D2 and vitamin D3 in either form (ie, 25-(OH) or 1,25-(OH)) in
a single test. 25-(OH) vitamin D is the most abundant metabolite of vitamin D, and it has a
long half-life. It is the component measured in most immunoassays for vitamin D. In contrast,
1,25-(OH)2 vitamin D has a much lower serum concentration, and a shorter half-life (4-6 hours).
PTH-related Protein (PTHrP)

This protein, nearly twice the size of PTH, is equipotent with PTH in inducing hypercalcemia.
PTHrP is secreted by numerous tumor tissues. Its shared homology allows PTHrP to bind PTH
receptors and stimulate renal proximal tubular reabsorption of calcium. The assay to measure
PTHrP shows less than 1% cross-reactivity with PTH.
Bone Markers
Markers for bone turnover can be classified into 2 groups, markers of bone formation and mark-
ers for bone resorption. Bone markers should not be used as definitive tests for the diagnosis
of osteoporosis. Their primary utility is to monitor response to treatment for bone disease. The
markers with the most clinical utility are described below.
Bone Formation Markers

Alkaline Phosphatase. This enzyme is present in a wide variety of tissues, one of which is the
bone. Most laboratory assays for alkaline phosphatase (ALP) measure total ALP. The bone-derived
fraction of ALP can be differentiated from its isoenzymes in serum by bone-specific ALP immu-
noassay or based on its instability. Bone ALP is denatured by heat and urea. Falsely elevated
results are commonly seen in liver disease.
Osteocalcin. Serum osteocalcin is a moderately specific marker for bone formation. Serum con-
centrations are highest in adolescence and in the newborn, when bone growth is most active,
and in renal failure due to clearance impairment. The serum osteocalcin concentration rises in
women from the 4th to the 10th decade as the bone turnover increases. Menopause induces a
marked increase in bone turnover, often with an increase in serum osteocalcin. Although not
as sensitive as collagen markers, measurement of osteocalcin can help predict bone loss in post-
menopausal women.
Procollagen Type I Intact N-terminal Propeptide (PINP). PINP, which is formed during col-
lagen synthesis, is the most sensitive marker of bone formation. Measurement of PINP by radio-
immunoassay in serum is recommended for monitoring of therapy to bone disease. It should be
measured prior to initiation of therapy and then subsequently 3 to 6 months later. PINP exhibits
less intraindividual biovariability than other collagen markers.
Bone Resorption Markers

N- and C-terminal Telopeptide of Type 1 Collagen (NTx and CTx). NTx and CTx are peptide
fragments formed during bone resorption through proteolytic processing of the N- and C-terminal
ends of type I collagen, respectively. These can be measured by immunoassay in both serum and
urine to assess response to treatment of bone disease. Significant intraindividual variability exists
in CTx concentrations because it is affected by diet, exercise, and time of day. NTx should be mea-
sured prior to initiation of therapy and then 3 to 6 months later to assess bone disease status.
Pyridinium Cross-links. Pyridinium cross-links, including deoxypyridinoline (DPD), are a
group of products formed during bone resorption as a result of collagen breakdown. These can
be measured by immunoassay and are useful in monitoring therapy. Urine pyridinium cross-
link concentrations can determine efficacy of bone disease treatment after as little as 2 months
of therapy.
Primary Hyperparathyroidism
Description
In primary hyperparathyroidism, there is excess secretion of PTH in the absence of an appro-
priate stimulus. The disease affects women about twice as frequently as it affects men, and the
incidence increases with age. The majority of cases of primary hyperparathyroidism result from
a single parathyroid adenoma, with hyperplasia of the parathyroids and parathyroid carcinoma
being less common causes. The hypercalcemia in hyperparathyroidism occurs as a result of the
direct action of PTH to increase resorption of bone calcium, PTH-induced renal tubular reab-
sorption of calcium, and synthesis of 1,25-(OH)2D3 that promotes the intestinal absorption of
calcium. Primary hyperparathyroidism is often identified in asymptomatic individuals who have The majority of
an unexpected serum hypercalcemia. Symptomatic patients with primary hyperparathyroidism cases of primary
may present with kidney stones, hypertension, polyuria, chronic constipation, depression, neuro- hyperparathyroidism
muscular dysfunction, recurrent pancreatitis, peptic ulcer, or an unexplained osteopenia. result from a single
parathyroid adenoma,
with hyperplasia
Diagnosis
of the parathyroids
Primary hyperparathyroidism may be suspected in patients with an isolated elevated total cal- and parathyroid
cium. In order to rule out effects of binding proteins, ionized calcium should also be determined, carcinoma being less
especially in patients with abnormal serum concentrations of total protein or albumin. As noted common causes. In the
earlier, the total serum calcium is 45% to 50% ionized, 40% to 45% protein-bound (mostly to diagnosis of primary
albumin), and 10% to 15% complexed with small inorganic and organic ions. On demonstration hyperparathyroidism, the
of hypercalcemia, serum PTH and fasting serum phosphorus (because the phosphorus concen- total serum calcium is the
tration in the serum is altered by diet) should be measured. Assays for PTH can measure the initial test.
intact molecule, carboxy-terminal, or midregion segments. The use of the intact PTH assay is
preferred, especially in patients with renal disease because PTH carboxy-terminal fragments can
TABLE 22–7 Laboratory Evaluation for Hyperparathyroidism and Hypoparathyroidism

Primary Secondary Hypercalcemia of
Laboratory Test Hyperparathyroidism Hyperparathyroidism Hypoparathyroidism Malignancy
Serum total or ionized Elevated Low or normal Low Elevated

calcium
Serum intact PTH Elevated Elevated Low or undetectable Low or normal
Serum PTH-related protein Undetectable Undetectable Undetectable Detectable in some

cancers
Serum 1,25-dihydroxy May be elevated but May be elevated, normal, Low Low or normal
vitamin D not usually required for or low depending on the
diagnosis blood concentrations of
calcium and phosphorus
Serum phosphorus Low or normal Normal Elevated Low or normal

(inorganic)
PTH, parathyroid hormone.
accumulate with decreased renal function. The diagnosis of hyperparathyroidism is made when
both persistent hypercalcemia and elevated serum PTH level are demonstrated. The serum inor-
ganic phosphorus may be low or normal in patients with primary hyperparathyroidism. Patients
with severe hyperparathyroidism can have bone pain, skeletal deformities, and even bone frac-
tures (Table 22–7).
Secondary Hyperparathyroidism
Description
Secondary hyperparathyroidism occurs when there is chronic hypocalcemia and an excessive
compensatory secretion of PTH. Chronic hypocalcemia is often a result of vitamin D deficiency
or renal disease with calcium losses into the urine. Inadequate dietary intake of calcium is a rare
cause of hypocalcemia. Secondary hyperparathyroidism is often associated with bone disease due
to PTH-mediated bone resorption and calcium release.
Diagnosis
In secondary hyperparathyroidism, there is an elevation in the PTH, but unlike primary hyper-
parathyroidism, the total and ionized calcium in the serum is low or normal. Tests to identify
causes of primary hyperparathyroidism, such as a parathyroid adenoma, should have negative
results. Tests should be performed to identify the cause of the chronic hypocalcemia leading to
secondary hyperparathyroidism. Vitamin D deficiency and renal disease, as noted previously, are
the most common causes of chronic hypocalcemia, and these may be diagnosed with the appro-
priate laboratory assays (Table 22–7).
Hypoparathyroidism Hypoparathyroidism
occurs most frequently Description
with unintentional
removal of the
Hypoparathyroidism occurs most frequently with unintentional removal of the parathyroid
parathyroids in the glands in the surgical excision of the thyroid gland. Other causes of hypoparathyroidism are
surgical excision of much less common. Hypocalcemia resulting from the hypoparathyroidism produces characteris-
the thyroid gland. In tic signs and symptoms, including numbness and tingling, and for patients with very low serum
hypoparathyroidism, the calcium levels, convulsions, and muscle spasms.
total and ionized calcium
levels in the serum Diagnosis
are low, with a low or In hypoparathyroidism, serum total and ionized calcium concentrations are low, with a low or
undetectable serum PTH undetectable serum PTH concentration. There is an elevation in the serum inorganic phosphorus
concentration.
associated with the decrease in serum calcium (Table 22–7).
Pseudohypoparathyroidism
Description
As the name implies, patients with pseudohypoparathyroidism have signs and symptoms that are
characteristic of hypoparathyroidism. This disorder results from a resistance of the tissues to the
action of PTH and not a PTH deficiency, hence the use of the term “pseudo.”
Diagnosis
Pseudohypoparathyroidism can be distinguished from true hypoparathyroidism by the high con-
centration of serum PTH, in the presence of a low serum calcium concentration, in patients with
the signs and symptoms of hypoparathyroidism. In addition, patients with pseudohypoparathy-
roidism demonstrate a lack of metabolic response when infused with PTH.
Vitamin D Deficiency
Description
Vitamin D deficiency, a major cause of secondary hyperparathyroidism and hypocalcemia, is
caused by insufficient sun exposure, decreased intestinal absorption, insufficient intake, renal or
liver failure, and numerous genetic disorders with defects in vitamin D processing, receptors,
or binding proteins. Vitamin D deficiency has recently been defined by the Institute of Medicine
as total 25-(OH) vitamin D <20 ng/mL (50 nmol/L), but this cutoff is assay-dependent. Based on
this cutoff, it is estimated that the prevalence of deficiency ranges from 20% to 100% of the elderly
population, and varies among younger individuals by race, age, and sun exposure. Severe vita-
min D deficiency in young children results in characteristic skeletal deformities known as rick-
ets. Consistent with secondary hyperparathyroidism, patients with vitamin D deficiency develop
bone disease often manifesting in osteomalacia, osteopenia, or osteoporosis. Vitamin D may also
have a role in numerous other tissues. Evidence suggests that 1,25-(OH)2 vitamin D may play a
direct or indirect role in immune modulation, blood pressure regulation, insulin production, and
cardiac muscle contractility. Vitamin D deficiency may be associated with colon, prostate, and
breast cancer, autoimmune disease, diabetes, and cardiovascular disease.
Diagnosis
High-risk patients (pregnant women, elderly, or patients with darker skin pigmentation) should
be screened for vitamin D deficiency by measurement of total 25-(OH) vitamin D. Concentra-
tions less than 20 ng/mL or a different lab-defined cutoff indicate deficiency. It is not useful to
measure 1,25-(OH)2 vitamin D in suspected vitamin D deficiency because of its short half-life and
tight regulation by numerous molecules. It is useful in the evaluation of patients with rare forms
of inherited rickets. The cause, prognosis, and treatment strategies for vitamin D deficiency can
be determined by also measuring PTH, magnesium, and phosphorous in plasma.
Hypercalcemia of Malignancy
Description
The most common cause of severe hypercalcemia in an inpatient hospital population is malig-
nancy. Tumors most often associated with hypercalcemia of malignancy include breast carci-
noma, multiple myeloma, and lung carcinoma. The serum calcium level may be elevated as a
result of osteolysis in the bone from metastases or humoral-induced hypercalcemia. In humoral The most common cause
hypercalcemia of malignancy, tumor production of PTHrP stimulates the PTH receptors to of severe hypercalcemia
induce hypercalcemia. The elevated calcium signals downregulation of PTH. The assay for PTHrP in an inpatient
is potentially useful when malignancy is suspected as a cause of hypercalcemia. hospital population is
malignancy. Tumors
most often associated
Diagnosis with hypercalcemia of
Hypercalcemia of malignancy must be differentiated from hyperparathyroidism. Patients with malignancy include breast
hypercalcemia of malignancy will have elevated total and ionized serum calcium, in the presence carcinoma, multiple
of suppressed or low PTH. The low PTH value is the differentiating feature of hypercalcemia of myeloma, and lung
malignancy from primary and secondary hyperparathyroidism, which are associated with high carcinoma.
concentrations of serum PTH (Table 22–7). For patients in whom humorally induced hypercal-
cemia is suspected, the most specific confirmatory test is the assay for PTHrP.
Hypocalciuric Hypercalcemia
Description
Familial hypocalciuric hypercalcemia (FHH) is a rare familial disease most often associated with
loss of function mutations in the calcium-sensing receptor gene product expressed in the para-
thyroid glands and kidneys. Normally, this receptor inhibits release of PTH from the parathyroid
glands in the presence of high calcium. In the absence of a functioning receptor PTH release is
uncontrolled, leading to elevated PTH and hypercalcemia. In the renal tubules, the calcium-sens-
ing receptors inhibit calcium reabsorption in the presence of high calcium. Without this receptor,
calcium is continuously reabsorbed and not excreted, accentuating the high serum concentra-
tions and leading to low urine calcium concentrations (hypocalciuria). Patients who are hetero-
zygous for this mutation typically have asymptomatic hypercalcemia, while those with 2 deficient
calcium-sensing receptor genes may require parathyroidectomy in infancy.
Diagnosis
Although rare, FHH is important because there is significant overlap in clinical and biochemical
parameters with primary hyperparathyroidism. Thus, it is recommended that the diagnostic eval-
uation include a combination of clinical, biochemical, and genetic tests. Clinically, patients with
FHH are usually asymptomatic, while those with primary hyperparathyroidism have symptoms
associated with elevated calcium as well as decreased bone density. FHH patients usually have a
personal and family history of hypercalcemia while those with hyperparathyroidism may not. The
laboratory workup for FHH shows elevated serum calcium and PTH, and usually a reduced urine
calcium. There is variability in urine calcium concentrations. Therefore, the calcium:creatinine
clearance ratio (CCCR) is the recommended test for identifying FHH. A CCCR <0.01 suggests
FHH, while a ratio >0.02 likely represents primary hyperparathyroidism. For patients with a
CCCR between 0.01 and 0.02, genetic identification of mutations in the calcium-sensing receptor
gene confirms the diagnosis of FHH.
Osteoporosis
Description
Osteoporosis is the most common metabolic disease of the bone associated with decreased bone
mass. The causes of osteoporosis are many and varied. Osteoporosis may be primary or second-
ary. It can occur in association with hyperparathyroidism as described before, as well as with
Cushing syndrome, acromegaly, prolonged use of heparin, excess vitamin D intake, and immobi-
lization, among other conditions and disorders.
Bone mineral density (BMD) studies are preferred for diagnosis of primary osteoporosis.
BMD estimates obtained by imaging studies are compared with BMD in normal populations to
generate a T-score. The WHO defines osteoporosis as a T-score ≤−2.5. T-scores between −1.0 and
−2.4 confirm osteopenia. Laboratory testing is preferred for the evaluation of secondary disease.
Bone turnover markers can be used to monitor treatment.
Diagnosis
Osteoporosis as a primary disorder is inferred by the absence of another disease known to
induce osteoporosis. Primary osteoporosis is generally idiopathic, postmenopausal, or senile.
Secondary osteoporosis is established by demonstrating an underlying process or treatment that
leads to osteoporosis.
Osteomalacia
Description
Osteomalacia is deficient mineralization of bone that results from disturbances in calcium and phos-
phorus metabolism. It can result from a nutritional deficiency of vitamin D, defects in vitamin D
metabolism or action, defects in mineral metabolism, or disturbances of the bone cells in the bone
matrix. When osteomalacia occurs before the cessation of growth, it is known as rickets. Skeletal
deformities appear in rickets because of the compensatory overgrowth of epiphyseal cartilage.
Diagnosis
Radiographic studies can demonstrate the disorder. The specific cause for osteomalacia, if it is
identified, is generally established with laboratory testing. There are many disorders associated
with the decreased mineralization of the bone.
Osteitis Deformans (Also Known as Paget Disease of Bone)

Description
Osteitis deformans is associated with osteoclastic resorption of bone and extensive production of
abnormal, poorly mineralized osteoid. This results in a bone that is structurally weak and prone
to deformity and fracture. The disorder may involve 1 bone or may be more generalized.
Diagnosis
In osteitis deformans, ALP is significantly elevated, which reflects osteoblastic proliferation in
the deformed bone. The serum calcium and inorganic phosphorus concentrations are usually
normal, but may be increased in some patients.
TESTES AND OVARIES

Male Physiology and Biochemistry
The male testes serve 2 important functions (Figure 22–9). One is the production of sperm, and
the other is the synthesis and secretion of androgens. Sertoli cells within the testes secrete inhibin,
and this glycoprotein inhibits the pituitary secretion of follicle-stimulating hormone (FSH). FSH
acts on the Sertoli cells to stimulate sperm production and the synthesis of inhibin. Leydig cells
in the testes are responsible for the production of androgens. The Leydig cells in the testes receive
stimulation by luteinizing hormone (LH) to promote the conversion of cholesterol, through many
intermediates, to testosterone. Testosterone, one of the androgens, is important for maturation of
sperm, production of male secondary sex characteristics, and providing negative feedback to the
anterior pituitary and hypothalamus to reduce the stimulation of the male testes. The hormone The androgens are a
secreted by the hypothalamus in the hypothalamic–pituitary–gonadal axis is gonadotropin- collection of 19 carbon
releasing hormone (GnRH). GnRH stimulates the release of both LH and FSH from the pituitary steroids that produce
in pulsatile patterns. Higher values for LH and FSH are found in the early morning hours. masculinization and
The androgens are a collection of 19 carbon steroids that produce masculinization and male male secondary sex
secondary sex characteristics. The main androgen secreted by the Leydig cells of the testes is characteristics. The main
testosterone. Other androgens secreted by the testes include androstenedione and DHEA. These androgen secreted by
compounds can be metabolized to testosterone and dihydrotestosterone (DHT) in target tissues. the Leydig cells of the
testes is testosterone.
Circulating testosterone is a precursor to DHT. As previously noted, a number of androgens are
Other androgens secreted
secreted by the adrenal glands, including DHEA, DHEA-sulfate (DHEA-S), androstenedione,
by the testes include
and androstenediol. Women also produce testosterone, but only 5% to 10% as much as men.
androstenedione and
Testosterone, as well as androstenedione, can be converted to estrogens. In men approximately DHEA.
6% to 8% of the testosterone is converted to DHT, but only about 0.3% to estradiol. Most of the
testosterone and DHT in the plasma is bound to plasma proteins. Only approximately 3% is free.
The 2 major proteins that bind testosterone and DHT are sex hormone-binding globulin (SHBG)
and albumin. In men, approximately 45% to 65% of protein-bound testosterone is associated with
SHBG and 35% to 50% is bound to albumin. Protein-bound testosterone in women is distrib-
uted approximately two thirds on SHBG and one third on albumin. The bioavailable testosterone
includes the small fraction that is free and the portion that is weakly bound to albumin. Testoster-
one binds less efficiently to albumin, and therefore it is available for tissue uptake when associated
with this protein. The main excretory metabolites of testosterone, androstenedione, and DHEA
are collectively known as 17-ketosteroids that can be quantitated in the urine.
[–] [–]
Hypothalamus
GnRH
[+]
[–] [–]
Pituitary
FSH LH
[+] [+]
Male Female
Testes Ovaries
Testes
Leydig cells Sertoli cells Leydig
Granulosa
cells Sertoli
cells cells
[+]
Inhibins
Testosterone Estradiol
FIGURE 22–9 The hypothalamic–pituitary–gonadal axis in males and females. [+] Stimulation;
[−] inhibition.
Laboratory Tests
Total Testosterone
Total testosterone measured by immunoassay is a commonly used first-line test in evaluating a
suspected gonadal dysfunction in adult males. Total serum testosterone represents both protein-
bound and non-protein-bound testosterone. Because it is subject to diurnal variation, testoster-
one should be measured in the morning.
Total testosterone Free and Weakly Bound Testosterone

measured by This is the bioavailable pool of circulating testosterone. In cases where total testosterone is
immunoassay is a abnormal, free testosterone can be assessed as a part of a panel that determines bioavailable
commonly used first-line testosterone and SHBG. Testosterone and SHBG are measured by immunoassay, and concen-
test in evaluating a male trations of free and bioavailable testosterone are derived from a mathematical equation based
for gonadal dysfunction. on the constants for the binding of testosterone to albumin and/or SHBG. This assay is not
recommended for women and children because the testosterone concentrations are much
lower. Therefore, for women and children, testosterone and SHBG are measured by a more
complex method, tandem mass spectrometry, and again free and bioavailable testosterone are
determined by the same mathematical equation.
SHBG can be measured by immunoassay separately, but the utility is largely in evalu-
ating bioavailable testosterone in men with suspected hypogonadism and women with
hyperandrogenism.
Testosterone Precursors and Metabolites

Immunoassays are available for quantitation of DHT, androstenedione, DHEA, DHEA-S,
and other related compounds. Large panel testing for adrenal and gonadal steroids measured
by LC/MS/MS are also available, especially for the evaluation of suspected congenital adrenal
hyperplasia.
DHEA and DHEA-S

Serum concentrations of DHEA and DHEA-S provide an assessment of adrenal androgen pro-
duction, which may be altered in patients with various conditions, including adrenal hyperplasia,
adrenal tumors, delayed puberty, and hirsutism. DHEA is almost entirely derived from the adre-
nal glands, and DHEA-S in the circulation originates mostly from the adrenal glands, although in
men some of it is derived from the testes. DHEA-S is the preferred test for assessing a suspected
adrenal androgen abnormality because addition of the sulfate group stabilizes DHEA, leading to
higher concentrations and a longer half-life in circulation.
Urinary 17-Ketosteroids
As noted above, the 17-ketosteroids in the urine are a collection of metabolites of androgenic
steroids secreted by the testes, the adrenal glands, and, in women, the ovaries. The urine 17-keto-
steroid test detects androsterone, DHEA, and several other steroids. However, it does not detect
cortisol, estrogens, pregnanediol, testosterone, and DHT because they do not have a ketone func-
tional group. In men, approximately 33% of the urinary 17-ketosteroids represent metabolites of
testosterone secreted by the testes, and most of the remaining 17-ketosteroids are derived from
steroids generated in the adrenal glands. In women, the 17-ketosteroids are derived almost exclu-
sively from androgens generated in the adrenal glands. The main purpose of measuring these
steroid metabolites is to assess androgen production by the adrenal gland. However, in men, a
decrease in 17-ketosteroids in the urine may result also from decreased production of testosterone
by the testes. Although the urine 17-ketosteroids are sometimes ordered to evaluate male andro-
genic status, this test does not detect the major androgens—testosterone and DHT. Therefore, if
low androgens are suspected, serum testosterone is the preferred test, rather than 17-ketosteroids.
Many clinicians now prefer the assessment of serum DHEA-S over urinary 17-ketosteroids for
investigation of adrenal androgen production because a 24-hour urine collection is not required
and many drugs interfere with the measurement of 17-ketosteroids.
Disorders Affecting Male Reproduction

Hypogonadotropic Hypogonadism. Hypogonadotropic hypogonadism in males is associated
with absent or decreased function of the testes. If this impairment is manifested early in life, sexual
development is retarded. In hypogonadotropic hypogonadism, there is a defect in the hypothalamus
or pituitary that reduces normal gonadal stimulation. There are many causes for this abnormality,
including panhypopituitarism and GnRH deficiency. A deficiency of GnRH in the hypothalamus
is responsible for the most common form of hypogonadotropic hypogonadism, Kallmann syn-
drome. Hypogonadism in men is diagnosed by at least 2 measurements showing of decreased total
testosterone in serum with symptoms of androgen deficiency. If total testosterone results are bor-
derline, hypogonadism can be confirmed by measuring free or bioavailable testosterone. Patients Patients with androgen
with hypogonadotropic hypogonadism have low testosterone and below normal or inappropriately insensitivity syndrome
normal serum concentrations of LH and FSH. Because there are many causes for the disorder, there (AIS), as it is now called,
is much heterogeneity in the severity of these hormonal deficiencies. A clinical picture of sexual have a severe defect
infantilism and low levels of LH, FSH, and testosterone in the serum are characteristic features of in androgen action,
hypogonadotropic hypogonadism. In order to differentiate pituitary or hypothalamic sources of with resistance to the
this disease, prolactin or other measures of pituitary function or imaging studies may be helpful. masculinizing effect of the
androgenic hormones.
Hypergonadotropic Hypogonadism. This disorder results from a defect in the testes, which This results in a female
may be a result of injury to the testes. There is active stimulation of the testes, but they are unre- habitus, with breast tissue
sponsive in this disorder. Apart from testicular injury, getting older is among the commonly and a vagina that ends
encountered causes of hypergonadotropic hypogonadism. The disorder can also result from tes- in a blind pouch, and
ticular damage from radiation or chemotherapy. undescended male testes.
TABLE 22–8 Laboratory Evaluation for Males With Hypogonadism and Complete
Androgen Insensitivity Syndrome (AIS)
Disorder Laboratory Test Results for LH, FSH, and Testosterone
Hypogonadotropic hypogonadism Low serum concentrations of LH, FSH, and testosterone
Hypergonadotropic hypogonadism Elevated serum concentrations of LH and FSH with a low serum
concentration of testosterone
Testicular feminization syndrome Elevated or occasionally normal serum testosterone for a male, with
an elevated serum LH
Patients with hypergonadotropic hypogonadism have elevated concentrations of LH and FSH

in the presence of decreased levels of testosterone. When the source of the gonadal failure is unclear,
karyotyping may identify chromosomal anomalies as the cause of the testicular abnormality.
Androgen Insensitivity Syndrome (Testicular Feminization Syndrome). Patients with
androgen insensitivity syndrome (AIS), as it is now called, have a severe defect in androgen
action, with resistance to the masculinizing effect of the androgenic hormones. This results in
a female habitus, with breast tissue and a vagina that ends in a blind pouch, and undescended
male testes.
The circulating concentration of testosterone in patients with AIS (Table 22–8) is normal
or elevated for a male. An elevation in testosterone can result in estrogen formation in these
individuals because testosterone is a precursor for estrogen. The serum concentration of LH is
increased, presumably because of resistance to the negative feedback of testosterone within the
pituitary and hypothalamus.
Erectile Dysfunction (Formerly Impotence). There are many causes for the persistent inabil-
ity to develop or maintain a penile erection sufficient for intercourse and ejaculation. Although
psychogenic impotence is the most common (up to 50%), there are many endocrinologic and
nonendocrinologic disorders that are associated with impotence. These include vascular disease,
diabetes mellitus, hypertension, neoplasms, and adverse drug effects.
An endocrinologic study of the patient may be pursued by measuring the serum testosterone
in the early morning, along with LH and FSH, to assess the hypothalamic–pituitary–male gonadal
There are many causes axis for testosterone production. Chapter 19 has additional discussion of this topic.
for the persistent inability
to develop or maintain a
penile erection sufficient Female Physiology and Biochemistry
for intercourse and The ovaries function to produce ova and secrete sex hormones, notably estrogens and progestins.
ejaculation. Although Estrogens maintain the female secondary sex characteristics. They are also essential in the regu-
psychogenic impotence lation of the menstrual cycle, and breast and uterine growth in the maintenance of pregnancy
is the most common (see Chapter 20 for a discussion on pregnancy). The estrogens have a major impact on calcium
(up to 50%), there are metabolism, and the estrogen depletion associated with menopause results in a loss of bone min-
many endocrinologic
eral content. Most of the estrogens in the body are secreted by the ovarian follicles and the corpus
and nonendocrinologic
luteum. During pregnancy, estrogen is also synthesized in the placenta. Only minute quantities
disorders that are
are synthesized by the adrenals. The normal human ovary produces estrogens, progestins, and
associated with
impotence. androgens, but the primary products are estradiol and progesterone. More than 20 different estro-
gens have been identified. Those with clinical importance are estradiol, also known as E2; estrone,
also denoted as E1; and estriol, that is E3. Estradiol is derived almost exclusively from the ovaries,
and for that reason the serum estradiol level is considered a reflection of ovarian function. In the
nonpregnant state, most of the estrogen (microgram quantities) is derived from the ovaries. In
pregnant women, the major source of estrogen is the placenta, which secretes estriol as the major
product in milligram amounts. Like most other steroid hormones, the vast majority of the circu-
lating estrogen is bound to plasma proteins. More than 95% of circulating estradiol is bound with
high affinity to SHBG and, less avidly, to albumin.
Progesterone is a female sex hormone in the progestin family that plays a central role in
female reproductive endocrinology. It is involved in regulation of the menstrual cycle and is
produced during pregnancy by the placenta. In the nonpregnant state, progesterone is produced
largely by the ovary. The adrenal cortex is only a minor source of progesterone production in both
sexes, and progesterone is made in very small quantities in the testes in men. More than 90% of
the progesterone is protein-bound in the circulation to corticosteroid-binding globulin. Proges-
terone can be metabolized to 3 groups of metabolites, one of which is the pregnanediols. Urinary
pregnanediol concentration can be used as an index of endogenous production of progesterone
because it correlates with alterations in progesterone synthesis and metabolism.
There is a tightly coordinated feedback system among the hypothalamus, anterior pituitary,
and ovaries in adolescent and adult women to regulate menstruation. Each menstrual cycle con-
sists of a follicular and a luteal phase. Day 1 is the first day of menstrual bleeding. The follicular
phase is associated with follicle growth and is the first part of the cycle. Ovulation occurs around
day 14 of the menstrual cycle, and the luteal phase follows in the last half of the cycle.
In general, follicular growth in the ovary is stimulated by FSH, and ovulation and progester-
one secretion from the developing corpus luteum are driven by LH. During the menstrual cycle:
• FSH increases during the early part of the follicular phase and then declines until ovulation; In general, follicular
there is a gradual decrease in FSH through the luteal phase. FSH guides selection of a growth in the ovary
dominant follicle. is stimulated by FSH,
• LH secretion increases around the middle of the follicular phase and just before ovulation; and ovulation and
estrogen secretion in the follicular phase stimulates the pituitary to release LH in a surge, progesterone secretion
with the peak value for LH appearing 10 to 12 hours before ovulation. from the developing
• Estradiol concentrations increase as the selected dominant follicle begins to secrete this corpus luteum are driven
hormone during midfollicular phase. Its concentrations rapidly rise as the follicle matures. by LH.
The estradiol concentration then falls abruptly just before ovulation. At ovulation the
ovum is released from the follicle. The leftover tissue, called the corpus luteum, is essential
for establishing early pregnancy. The corpus luteum secretes estradiol and progesterone
to facilitate implantation. If the ovum is not fertilized, the corpus luteum breaks down
and estradiol and progesterone synthesis decline after about 14 days. The decline in both
hormones signals the beginning of the menstrual cycle.
• Progesterone is at very low concentrations during the follicular phase; with the midcycle surge
of LH and ovulation, the corpus luteum secretes progesterone that increases and reaches its
peak concentration approximately 8 days after the midcycle LH surge. As the corpus luteum
degrades, progesterone concentrations decline to baseline levels at the end of the luteal phase.
Figure 22–9 illustrates the complex relationships in the hypothalamic–pituitary–female
gonadal axis.
Laboratory Tests
Estrogens
Serum estrogen levels are represented by the estradiol (E2) concentration, because estriol (E3) in
a nonpregnant woman is derived almost exclusively from estradiol. In addition, blood estrone
(E1) levels typically parallel estradiol levels throughout the menstrual cycle, but at a lower con-
centration. Urinary estrogens can be quantitated as total estrogens or as fractionated estrogens
with measurement of estradiol, estrone, and estriol. Since estradiol is derived primarily from the
ovaries, the estradiol concentration in the urine can provide a more accurate reflection of ovarian
function than total urinary estrogen.
Progesterone
The progesterone concentration in serum is a reflection of progesterone production. Assays for
urinary progesterone metabolites are used much less frequently to assess progesterone synthesis
than tests for serum progesterone.
Endocrinologic Disorders Affecting Female Reproduction

Healthy women display considerable variations in the length of the menstrual cycle, but most
women have cycles between 25 and 30 days in length (see Figure 22–10). The absence of men-
strual bleeding is known as amenorrhea. Primary amenorrhea refers to women who have never
150 30 300 30
Serum progesterone, ng/mL

Serum estradiol, pg/mL
Serum FSH, IU/L
Serum LH, IU/L
100 20 200 20
Estrogen
FSH
50 10 100 10
LH
Progesterone
0 0 0 0
−14 −7 0 7 14 −14 −7 0 7 14
Days from midcycle surge Days from midcycle surge
FIGURE 22–10 The changes in LH, FSH, estrogens, and progesterone in menstruating females.
Day 1: start of menses; days 5 to 7: estrogen secretion begins; day 13: LH surge; day 14: ovulation;
days 18 to 23: increased estrogen and progesterone from the corpus luteum; day 25: decreased
estrogen and progesterone with demise of the corpus luteum and breakdown of the endometrium.
menstruated, and secondary amenorrhea refers to women in their reproductive years in whom
menstruation was present and then ceased for at least 6 months.
Primary Amenorrhea. Primary amenorrhea is established if spontaneous regular menstruation
has not begun by the age of 16 years, with or without the presence of secondary sex characteris-
tics. The list of causes of primary amenorrhea is lengthy. They include lower genitourinary tract
Primary amenorrhea defects such as imperforate hymen; a host of ovarian disorders—approximately 40% of females
refers to women who have with primary amenorrhea have Turner syndrome (45 X karyotype) or pure gonadal dysgenesis
never menstruated, and (either a 46 XX or XY karyotype); adrenal disorders such as CAH; thyroid disorders, notably
secondary amenorrhea hypothyroidism; pituitary–hypothalamic disorders such as hypopituitarism and Kallmann syn-
refers to women in their drome (which also affects men); and pregnancy.
reproductive years in Because of the long list of possible causes, the workup for primary amenorrhea should begin
whom menstruation was with a careful history and physical examination to look for anatomic defects, development of
present and then ceased secondary sexual characteristics, and/or a personal or family history of short stature, infertility,
for at least 6 months.
and/or amenorrhea. The laboratory evaluation for amenorrhea begins with measurement of hCG
to rule out pregnancy. If not pregnant, patients should undergo imaging studies looking for a
uterus and gonads. Patients with a detectable uterus should be evaluated for hypothyroidism and
hyperprolactinemia (discussed below). High TSH and low free T4 results suggest the amenorrhea
is due to primary hypothyroidism. An elevated prolactin result should prompt a physician to
perform an MRI in search of a pituitary adenoma. Patients with normal TSH and prolactin should
be evaluated for gonadotropic function by measuring LH and FSH. Estrogen measurements may
be helpful to determine the cause of disease. Because of the day-to-day variability in estrogen
concentrations, the progestin challenge test may be helpful to establish estrogen reserves and/
or etiology of primary amenorrhea. Theoretically, if progesterone is given to an estrogen-primed
uterus, withdrawal bleeding (menstruation) will occur. Progesterone is given orally for up to
1 week. Bleeding should occur within 1 week of progesterone withdrawal if the woman’s ovaries
have produced enough estrogen (>40 pg/mL serum) to prime her uterus.
If the patient has congenital anomalies, a karyotype evaluation to look for cytogenetic abnor-
malities may be helpful (Table 22–9).
Secondary Amenorrhea. Secondary amenorrhea is more common than primary amenorrhea
and is the absence of regular menstruation for at least 6 months in a woman who has previously
had menses. Oligomenorrhea is present if a woman has less than 9 menstrual cycles per year.
The causes of secondary amenorrhea include many of those for primary amenorrhea. However,
there are a number of conditions associated with secondary amenorrhea that are independent of
primary amenorrhea. Most notably, pregnancy is a common cause of amenorrhea and must be
considered first in a patient who has stopped menses. An elevated prolactin concentration, which
may be induced by a prolactin-secreting tumor, can produce oligomenorrhea or amenorrhea,
TABLE 22–9 Laboratory Evaluation of Women With Amenorrhea

Disorder Associated Disorders and Potentially Relevant Laboratory Tests
Primary amenorrhea Pregnancy—test for hCG

Prolactin-secreting pituitary tumor—serum prolactin level
Turner syndrome and pure gonadal dysgenesis—LH and FSH measurements
and karyotype analysis
Congenital adrenal hyperplasia—adrenal hormone and metabolite measurements
Hypothyroidism—selected thyroid hormone assays
Hypopituitarism—pituitary hormone concentrations in serum
Secondary amenorrhea Pregnancy—test for hCG

Prolactin-secreting pituitary tumor—serum prolactin level
Polycystic ovary syndrome—serum testosterone (free or total), adrenal
androgens (DHEA-S), and appropriate radiographic studies
Cushing syndrome—see the section “Hyperfunction Involving Glucocorticoids
With or Without Mineralocorticoids: Cushing Syndrome”
Adult-onset congenital adrenal hyperplasia—adrenal hormone and metabolite
measurements (17-hydroxyprogesterone) as described in this chapter
Hypothyroidism and hypopituitarism—as described above for primary amenorrhea
hCG, human chorionic gonadotropin.
presumably by inhibition of the release of LH and FSH by the prolactin. Patients with secondary
amenorrhea can be divided into those with and without signs of hirsutism and androgen excess.
Hirsutism is the excessive growth of terminal hair in women and in children, in a distribution sim-
ilar to that which occurs in postpubertal men. Causes of hirsutism may be androgen-dependent,
with abnormalities often originating in the ovary or the adrenal gland, or androgen-independent,
sometimes from antiepileptic medications. Adult women with hirsutism and androgen excess
may carry a diagnosis of adult-onset CAH, ACTH-dependent Cushing syndrome, or polycystic
ovary syndrome, among other causes.
Because the list of disorders associated with secondary amenorrhea is even longer than the
list associated with primary amenorrhea (Table 22–9), the initial evaluation is very broad until the
differential diagnosis is narrowed by the results of physical examination, history, and initial radio-
graphic and laboratory studies. The laboratory workup for secondary amenorrhea begins with
measurement of hCG to rule out pregnancy. Hypothyroidism and hyperprolactinemia should
next be ruled out as the cause of disease by measuring TSH and prolactin. Patients with normal
TSH and prolactin should be evaluated for gonadotropic function by measuring LH and FSH.
Estrogen or a progestin stimulation test may be helpful in cases where the cause of amenor-
rhea is unclear. In women with hirsutism or other signs of hyperandrogenism, a total or free tes-
tosterone and DHEA-S should be measured. An elevation of DHEA-S points to an adrenal origin
as a cause for the hirsutism. An elevation in testosterone suggests either an adrenal or an ovarian
source, or an androgen-secreting tumor outside the adrenal and ovaries.
DISORDERS RELATED TO THE PITUITARY GLAND

GH has secretory spikes,
Growth Hormone/Anterior Pituitary with a half-life of about
20 minutes, which
typically occur several
Growth hormone (GH) is a major product of the pituitary gland. It is a single-chain polypeptide hours after meals and
that has structural similarities to prolactin and placental hormones, known as placental lactogens, exercise. The secretion
with which it has overlapping biological activities. GH has secretory spikes, with a half-life of about of GH also rises after the
20 minutes, which typically occur several hours after meals and exercise. The secretion of GH also onset of sleep and reaches
rises after the onset of sleep and reaches a peak in deepest sleep. Two hypothalamic factors control a peak in deepest sleep.
Exercise, stress, sleep,

low glucose
[+] [–] For GH-releasing hormone (GHRH)

[–] [+] For somatostatin release
Hypothalamus
[–] Growth hormone- Somatostatin

releasing hormone
(GHRH)
[+] [–]
[–]
Pituitary
Growth hormone
[+]
Insulin-like growth
factors (IGFs)
Liver and other tissues such
synthesized in liver
as cartilage and bone
and released into
the circulation
FIGURE 22–11 The regulation of growth hormone (GH) secretion. [+] Stimulation; [−] inhibition.
the release of GH from the pituitary. Growth hormone-releasing hormone (GHRH) stimulates
GH release from the pituitary, and somatostatin (also known as growth hormone-inhibitory hor-
mone [GHIH]) inhibits GH release. The larger influence on the release of GH by the pituitary is
the inhibitory action of somatostatin. To promote growth, GH in the circulation binds to target
tissues, mostly cartilage, bone, and other soft tissues. GH predominantly exerts its growth effects
by stimulating insulin-like growth factors (IGFs) that are produced in the liver and other tissues.
Because of its homology to insulin, GH directly affects lipid, carbohydrate, and protein metabo-
lism. IGFs, previously known as somatomedins, also have multiple effects on growth promotion
and metabolism. There are a number of IGFs. Unlike most other peptide hormones, IGFs circu-
late in the blood in a complex with plasma-binding proteins, known as insulin-like growth factor-
binding proteins (IGFBPs). The complex physiology of GH signaling is shown in Figure 22–11.
Laboratory Tests
Growth Hormone
Most of the assays for GH are performed using serum, because the concentration of GH in urine
is approximately 0.1% of that in serum. Human GH exists in the pituitary gland and in the cir-
culation as a heterogeneous mixture of isoforms. The presence of GH variants in serum can lead
to discrepant results among the different assays for GH, although most commercially available
GH assays are now calibrated against WHO-standardized materials. Even if the assay problems
Provocative tests aimed did not exist, a single random GH measurement is not usually clinically informative because of
at testing its stimulation the diurnal variability and pulsatile secretion of GH by the pituitary gland. Serum concentra-
or suppression are usually tions between pulses in healthy individuals are extremely low and may not even be detectable.
required to establish GH Provocative tests aimed at testing its stimulation or suppression are usually required to establish
abnormalities. GH abnormalities (Table 22–10). A commonly used stimulation test to assess the adequacy of
GH secretion is the insulin tolerance test, which produces a transient hypoglycemia to provoke
TABLE 22–10 Laboratory Evaluation for Growth Hormone Abnormalities

Laboratory Test Growth Hormone Excess Growth Hormone Deficiency
Serum growth Single measurements of GH are not often reliable Because GH may be low
hormone (GH) because GH secretion is episodic and diurnal and other in both normal children
conditions can increase GH secretion; normal individuals and GH-deficient patients,
have markedly suppressed GH concentrations after it is necessary to show an
administration of an oral glucose load, but patients inadequate rise of serum GH
with acromegaly/gigantism do not have suppressed in response to 2 different
GH concentrations in response to same challenge provocative stimuli
Serum IGF-1 IGF-1 is elevated in nearly all patients with Low in most deficient patients,
acromegaly/gigantism but low in many other clinical
conditions as well
Radiology Sellar enlargement in 90% of cases; if present, a pituitary

tumor should be localized
GH release. One GH suppression test involves the ingestion of an oral glucose load, which in
healthy individuals suppresses GH secretion from the pituitary.
Insulin-like Growth Factors

IGF-1 circulates in much higher concentrations in plasma than GH, and its secretion is not epi-
sodic or diurnal. Therefore, IGF-1 is a good screening test of suspected GH abnormalities and
for monitoring therapy in patients with known abnormalities. A single elevated IGF-1 in patients
with signs and symptoms of GH excess should be followed with an oral glucose tolerance test (GH
suppression testing). A single decreased IGF-1 should prompt treatment in patients with known
growth deficiency or an insulin tolerance (or other GH stimulation) test in patients suspected to
have GH deficiency. There are marked differences in IGF-1 concentration between adults and
children. Therefore, it is very important to establish age-specific reference intervals.
Growth Hormone Excess—Acromegaly and Gigantism

Description
The most common cause of excess GH production is a chromophobe adenoma of the pituitary
gland. A prolonged excess of GH results in an overgrowth of the skeleton with acral enlargements, as
well as overgrowth of the soft tissues, and these are found in 100% of the patients with this condition.
In adults, this condition is known as acromegaly. Because GH has an action on the cartilaginous por-
tion of the bone, GH excess in children before long bone growth is completed results in gigantism.
Diagnosis
The most important requirement for diagnosis of GH excess is a demonstration of unsuppressible
GH secretion. Because of the episodic secretion of GH, as many as 10% of patients with active
acromegaly have random serum GH concentrations that are within the normal reference interval. A prolonged excess of GH
An elevated IGF-1 measurement prompts provocative testing and correlates with disease severity. results in an overgrowth
Serum GH concentrations are typically not suppressed by oral glucose loading in patients with of the skeleton with acral
acromegaly. Their serum GH concentrations show either no change from baseline or a slight enlargements. In adults,
increase. Healthy individuals show marked suppression after oral glucose load. The serum IGF-1 this condition is known
concentration, even as a random test, correlates with the clinical severity of acromegaly better as acromegaly. GH excess
than the test for glucose-induced GH suppression. in children before long
bone growth is completed
results in gigantism.
Growth Hormone Deficiency
Description
A deficiency of GH may be congenital or acquired. Children who have inadequate GH produc-
tion or action will not grow to full height. It should be noted that growth retardation is typi-
cally not usually caused by GH deficiency. However, children with growth retardation or reduced
growth velocity with no obvious explanation should be evaluated for a GH deficiency. In children,
causes of GH deficiency include anatomic damage to the pituitary or hypothalamus, isolated GH

deficiency in the pituitary, and a combination of pituitary hormone deficiencies from a variety of
causes. In adults, the most common causes of GH deficiency are pituitary irradiation and a pitu-
itary adenoma that impairs GH secretion. Since adults are usually at full height, GH deficiency
does not change growth velocity. However, it is associated with cardiac abnormalities, increased
cholesterol, reduced muscle mass, energy, and bone density, and lean body mass. GH replacement
in deficient adults may increase lean body mass and bone density, and reduce cholesterol.
Diagnosis
After ruling out other causes of short stature, patients with signs and symptoms of GH deficiency
can be screened by measuring IGF-1; if low, the diagnosis of GH deficiency requires the dem-
onstration of a persistently low GH concentration in 2 different provocative stimulation tests
(Table 22–10). The provocations for GH release that can be used include vigorous exercise, deep
sleep, and treatment with glucagon, l-DOPA, insulin, or arginine. Insulin and glucagon are the
most commonly utilized agents.
Prolactin production Prolactin/Anterior Pituitary

is regulated by stimuli Physiology and Biochemistry
from the hypothalamus,
but unlike most other Prolactin is secreted by the anterior lobe of the pituitary gland. It is a polypeptide with 198 amino
anterior pituitary acid residues that structurally resembles GH, and is secreted by lactotrophic cells in the anterior
hormones, its release is pituitary. Prolactin production is regulated by stimuli from the hypothalamus, but unlike most
primarily controlled by other anterior pituitary hormones, its release is primarily controlled by inhibition rather than
inhibition rather than by by stimulation (see Figure 22–12). The primary negative stimulus to the pituitary that limits
stimulation. prolactin secretion is provided by dopamine. There are various stimulatory factors for prolactin
release, along with the inhibitory compounds. One stimulatory factor is TRH. Another mecha-
nism to increase prolactin release from the pituitary is to decrease the inhibitory effect of dopa-
mine. A number of medications inhibit dopamine action. Prolactin secretion, like that of several
other anterior pituitary hormones, is episodic. Concentrations of prolactin are at their lowest at
midday, with the highest values shortly after the onset of sleep. The major physiologic stimulus
Suckling & other stimuli
[+]
[–]
Hypothalamus
Drugs that inhibit [–]

Dopamine Prolactin releasing
dopamine action peptide, thyrotropin,
releasing hormone
[–] [+]
Drugs that have a [–] Pituitary
dopamine-like effect
Prolactin
[+]
Milk production
Breast
FIGURE 22–12 The regulation of prolactin secretion. [+] Stimulation; [−] inhibition.
for prolactin release is suckling of the breast in lactating women. This results in a rise in maternal
plasma prolactin concentrations within minutes to initiate milk production. Prolactin controls
the initiation and maintenance of lactation only if the breast tissue is appropriately primed by
estrogens and other hormones for ductal growth, development of the breast lobular and alveolar
system, and the synthesis of specific milk proteins.
Laboratory Tests
Prolactin
As with GH, there is molecular heterogeneity in prolactin with multiple isoforms, which can lead to
discrepant results among the immunoassays. A high-molecular-weight isoform, macroprolactin,
is a clinically inactive species that contains large aggregates of prolactin with immunoglobulins. It
is recognized by many commercial immunoassays and can be the source of hyperprolactinemia.
Physicians should check for macroprolactin by precipitation or size exchange chromatography in
any patient with an elevated serum prolactin of unknown origin. The best serum specimen is one
that is collected 3 to 4 hours after the subject has awakened. It is important to collect the speci-
men after an overnight fast, when the patient is still resting, because emotional stress, exercise,
ambulation, and protein ingestion all elevate the baseline prolactin level. Multiple sampling may
be necessary because of the episodic secretion of prolactin.
Hyperprolactinemia
Description. There are many causes for an elevated prolactin concentrations, one of which is a
pituitary adenoma. Pregnancy, chronic renal failure, chest wall trauma, primary hypothyroid-
ism, and a host of medications can also elevate prolactin levels. Elevation of the serum prolactin
concentration is associated with many different signs and symptoms. In women, these include
anovulation, with or without menstrual irregularity, and amenorrhea. Men with prolactin-
secreting pituitary adenomas often present with oligospermia, impotence, or both.
Diagnosis. A patient with a prolactin-secreting pituitary adenoma generally has a higher eleva-
tion of serum prolactin than someone with hyperprolactinemia from a different cause. Because
so many medications can provoke prolactin release, and medications are the most common cause
of an elevated serum prolactin level, a medication history is very important. Noteworthy medica-
tions that can elevate the serum prolactin level include estrogens, dopamine antagonists such as
haloperidol, histamine receptor-blocking agents such as cimetidine, and tricyclic antidepressants.
Hypoprolactinemia
Description and Diagnosis. This condition is not detected unless a woman fails to lactate post-
partum. A low prolactin level in a woman in this setting is consistent with hypoprolactinemia.
Antidiuretic Hormone (ADH)/Posterior Pituitary

The posterior pituitary secretes oxytocin and ADH, also known as arginine vasopressin. ADH is
a small peptide with 9 amino acids. Oxytocin has a similar structure. Release of hormones from
the posterior pituitary into the circulation occurs with stimulation of selected neurons. In the
circulation, ADH and oxytocin are usually not bound to carrier proteins.
ADH secretion is triggered by elevated plasma osmolality as well as decreased blood vol-
ume or blood pressure (Figure 22–13). Even a small increase in osmolality causes stimulation
of ADH release to increase water retention and decrease the plasma osmolality toward normal.
ADH induces increased permeability of water in the renal collecting ducts leading to increased
water reabsorption and concentration of urine. ADH is also known as vasopressin because it
binds to receptors on smooth muscle cells that induce vasoconstriction, thereby increasing the
blood pressure and volume. Other nonosmotic stimuli such as pain, stress, sleep, exercise, and
a variety of pharmacologic compounds induce ADH secretion from the posterior pituitary as
well. Negative feedback for ADH release is provided by atrial natriuretic peptide (ANP). With an
increased circulating blood volume or a decreased osmolality, ANP concentrations increase,
Posterior pituitary
[+] [–]
Increased osmolality of extracellular fluid Antidiuretic hormone (ADH)
[+]
Thirst Water conservation
Water intake
Increased circulating Decreased osmolality

volume of extracellular fluid
Increased atrial natriuretic peptide (ANP)
FIGURE 22–13 The regulation of circulating fluid volume and osmolality. [+] Stimulation;
[−] inhibition.
inducing decreased ADH release. The osmolality of the plasma also impacts the thirst center to
coordinate oral intake of water and conservation of water in the kidney.
Laboratory Tests
Antidiuretic Hormone
ADH can be measured using an immunoassay. It is a temperature-sensitive peptide, and plasma
testing should be performed within 24 hours after collection. Freezing the specimen stabilizes it
for many months. A single ADH measurement is not diagnostic, and the result should be assessed
in the context of the results of serum and urine osmolality testing.
Serum and Urine Osmolality

Serum and urine osmolality are measured using a freezing point depression osmometer. Serum
osmolality can be estimated by various mathematical formulas using sodium, glucose, and BUN
concentrations.
Water Deprivation Test

Patients with suspected diabetes insipidus (DI) are deprived of all fluids until urine osmolality is
constant for 3 hours and/or plasma osmolality is greater than the normal range. Once constant,
serum osmolality and ADH are measured. ADH is then administered and urine osmolality and
volume are measured at 1 and 2 hours post administration.
Polyuria can occur from Polyuria

3 main causes. The first is
Description. A deficiency of ADH or resistance to the action of ADH results in the failure of the
deficient production of
renal tubules to reabsorb water and, as a result, an increased amount of water is lost into the urine.
ADH, as occurs in central
Because urine output is dependent on fluid intake, a normal urine output cannot be defined.
diabetes insipidus. The
second cause for polyuria However, whenever there is more than 2.5 L of urine generated per day, an investigation for a
is deficient ADH action cause of polyuria is usually indicated.
in the kidney. The third Polyuria can occur from 3 main causes. The first is deficient production of ADH, as occurs
cause of a polyuric state is in central DI. In this disorder, the pituitary gland fails to secrete normal amounts of ADH in
excessive water intake. response to stimuli. When the thirst mechanism is normal, increased fluid intake compensates for
water lost into the urine and thereby prevents dehydration. Severe dehydration can occur if the
TABLE 22–11 Laboratory Evaluation for Disorders of Water Uptake and Excretion
Serum Sodium and Urine Sodium and
Baseline Disorder Osmolality Osmolality Plasma ADH
SIADH Low Normal-high High
Central diabetes insipidus Normal-high Low Low
Nephrogenic diabetes Normal-high Low Normal-high

insipidus
Psychogenic polydipsia Normal-low Low Normal-low

SIADH, syndrome of inappropriate antidiuretic hormone secretion.
thirst center is abnormal, and there is excess water loss into the urine. Congenital disorders of the
pituitary or neoplastic diseases, neurological surgery, head trauma, ischemia, and autoimmune
disorders account for most of the cases of central DI.
The second cause for polyuria is insensitivity of renal tubules to ADH. This is known as neph-
rogenic DI and it can be caused by any damage to the renal tubules that impairs water reabsorption.
The third cause of a polyuric state is excessive water intake. This is known as psychogenic or
primary polydipsia. In rare cases, hypothalamic disease can affect the thirst center and induce
polydipsia. There are also many medications that can affect the thirst center and cause polydipsia.
Diagnosis. The differential diagnosis of a polyuric state requires measurements of serum and
urine osmolality, serum sodium, urine volume, and plasma ADH concentrations. The first step
is to document that polyuria exists by establishing that the urine volume exceeds 2.5 L per day.
Glycosuria must be excluded as a cause of the polyuria, as hyperglycemia with diabetes mellitus is
a common cause of polyuria. Patients with central DI have hypernatremia, high plasma and low
urine osmolality, and low or inappropriately normal plasma ADH level because the pituitary is
unable to secrete ADH. Patients with nephrogenic DI also have a high serum sodium and osmo-
lality and a low urine osmolality with polyuria and polydipsia. Because of renal insensitivity, they
have a normal to high plasma ADH because the hypothalamus generates excess hormone in an
attempt to compensate for high plasma osmolality. Patients with primary polydipsia usually have
hyponatremia and normal serum and low urine osmolality, with an appropriately low-normal
plasma ADH concentration (Table 22–11). The diagnosis of DI can be confirmed with a pro-
vocative test called the water deprivation test. After dehydration, a urine osmolality greater than
plasma with a low ADH, followed by an increase in urine osmolality of more than 10% in 1 hour
following ADH administration, indicates central DI. The excess ADH promotes reabsorption of
water by the kidney, resulting in a decreased urine volume and an increased urine osmolality. A
high ADH after dehydration, followed by a failure to increase urine osmolality after ADH admin-
istration, suggests nephrogenic DI because the defect in this disorder is a failure of the kidney to
respond to ADH. After water deprivation, patients with primary polydipsia will have urine osmo-
lality higher than serum and show no increase in urine osmolality after ADH administration.
Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH) SIADH is an autonomous,

sustained synthesis
Description and release of ADH in
SIADH is an autonomous, sustained synthesis and release of ADH in the absence of stimuli. Thus, the absence of stimuli.
plasma ADH concentrations are inappropriately increased relative to the osmolality. There are Thus, plasma ADH
a number of known causes of SIADH; it is common among patients with pulmonary or central concentrations are
nervous system disorders. Another cause is production of ADH by a malignant tumor, especially inappropriately increased
a small cell carcinoma of the lung. In addition, there are a number of medications that stimulate relative to the osmolality.
the production of ADH. The patient’s blood volume is modestly expanded, and serum sodium
concentrations may be decreased along with serum osmolality. SIADH is a commonly encoun-
tered cause of hyponatremia in hospitalized patients.
Diagnosis
Patients with SIADH usually have a low serum osmolality, a urine osmolality greater than that
of serum, and an elevated urine sodium concentration (Table 22–11). There are many causes for
hyponatremia other than SIADH, including congestive heart failure, renal insufficiency, nephrotic
syndrome, liver cirrhosis, and treatment with medications that stimulate ADH secretion. SIADH
is not often assessed by measurement of plasma ADH because the ADH level is not usually neces-
sary to make the diagnosis.
NEOPLASTIC DISORDERS
Multiple Endocrine Neoplasia
Description
MEN is a syndrome most often inherited as an autosomal dominant trait. The MEN syndromes
are associated with hyperplasia or tumors in multiple endocrine glands at the same time. There
have been many recent advances in the understanding of the genetic basis of the different types
of MEN.
• Multiple endocrine neoplasia type-1 (MEN 1; Wermer syndrome)—MEN 1 syndrome
involves hyperplasia or neoplasms in 1 or more of the following: the parathyroid gland, the
pancreatic islet cells, or the anterior pituitary in patients with a known family history of
MEN 1. In the absence of a family history of the syndrome, however, at least 2 or more of
the primary MEN 1 tumor types must be involved for a diagnosis of MEN 1. The hormonal
presentation of MEN 1 is highly variable because the pituitary and the pancreatic islet
cells in neoplastic states can secrete many different hormones. Although the prevalence
is reported to be 20 to 200 per million live births, it is likely to be greatly underestimated
Multiple endocrine because the clinical expression of MEN 1 varies and often presents with mild symptoms.
neoplasia type-1 Patients with MEN 1 usually present in the fourth decade of life. MEN 1 has been linked to
(MEN 1) syndrome a gene mutation in the MEN 1 (menin) gene on chromosome 11.
involves hyperplasia or • MEN 2 (Sipple syndrome)—The most commonly found abnormality in the MEN 2
neoplasms in 1 or more syndrome is MTC that occurs in over 95% of patients with MEN 2. Pheochromocytoma
of the following: the develops in over 50% of patients with MEN 2, and parathyroid hyperplasia or adenoma
parathyroid gland, the produces hyperparathyroidism in 15% to 30% of patients with MEN 2. MEN 2 includes
pancreatic islet cells, or 3 major phenotypes. Over 90% of the cases of MEN 2 are MEN 2A that includes risk
the anterior pituitary. The
of developing MTC, pheochromocytoma, and hyperparathyroidism. The familial cases
most commonly found
of MEN 2A are most often diagnosed in the third or fourth decade of life. In MEN 2B,
abnormality in the MEN 2
parathyroid disease is rare and there are separate developmental abnormalities such as
syndrome is medullary
thyroid carcinoma that ganglioneuromatosis and marfanoid habitus, in addition to pheochromocytoma and MTC.
occurs in over 95% of MEN 2B generally presents 10 years earlier than MEN 2A. It accounts for approximately
patients with MEN 2. 5% of all MEN 2 cases. MEN 2B is usually recognized early in life. The child with MEN
2B has a characteristic facial appearance with a failure to thrive, mucosal neuromas, and
constipation or diarrhea due to the ganglioneuromatoses in the gut. The diagnosis can
be made conclusively by demonstrating the presence of a mutation in the RET proto-
oncogene. The RET proto-oncogene product is a receptor tyrosine kinase that transmits
growth and differentiation signals. The third form of MEN 2 is familial MTC in the absence
of pheochromocytoma and hyperparathyroidism. This disorder has a later onset than MEN
2A or MEN 2B and usually has a good prognosis. The most common clinical presentation
in the patient with medullary carcinoma is a mass in the neck. The diagnosis is most often
made by histopathologic review of a specimen acquired by fine needle biopsy.
Diagnosis
Because of the variety of hormonal abnormalities in MEN 1, many different assays are needed to
demonstrate hyperplasia or neoplasms of the parathyroid, pancreatic cells, and/or anterior pitu-
itary, all of which may be involved in MEN 1. Genetic mutations in the coding sequence of the
RET proto-oncogene are found in the vast majority of patients with MEN 2 (both MEN 2A and
MEN 2B) and those with isolated familial MTC. Any first-degree relative of a patient carrying an
MEN-associated mutation should also be evaluated. Onset of C-cell hyperplasia and malignancy
of the thyroid in patients with a known RET proto-oncogene mutation should be monitored by
measuring calcitonin and routine thyroid ultrasounds (see the section “Thyroid”).
Carcinoid Tumors
Description
Carcinoid tumors are the most common of the endocrine tumors. They are generally found in the
wall of the gastrointestinal tract, but also can be found in the pancreas, rectum, ovary, and lung.
Tumors originating from the primitive foregut include carcinoid of the bronchus, the stomach,
the first portion of the duodenum, and the pancreas. These tumors often secrete 5-hydroxytrypto-
phan, histamine, and other peptides. Carcinoid tumors originating from the primitive midgut are
those found in the second portion of the duodenum, the jejunum, the ileum, and the ascending
colon. These tumors secrete serotonin, also known as 5-hydroxytryptamine, and other peptides.
They are associated with the development of carcinoid syndrome, which is characterized by cuta-
neous flushing, gastrointestinal hypermotility with diarrhea, heart disease, bronchospasm, myop-
athy, and increased skin pigmentation. Tumors originating from the primitive hindgut include
those of the transverse colon, descending colon, and rectum. These tumors are typically silent
because they are usually nonsecretory. Therefore, functioning carcinoid tumors are more likely
to be detected if they secrete a compound that has biological activity. The serotonin-secreting
carcinoid tumors arising from the primitive midgut or foregut are the ones most often detected.
Silent carcinoid tumors are most often discovered incidentally at surgery for other disorders in
the gastrointestinal tract. Patients with silent carcinoid tumors may have vague abdominal pain
that is either undiagnosed or attributed to irritable bowel syndrome.
Diagnosis
Serotonin (5-hydroxytryptamine) is transported in the blood by platelets. It is metabolized to
5-hydroxyindoleacetic acid (5-HIAA). 5-HIAA is quantitatively the principal metabolite of sero-
tonin, and the majority of it is excreted into the urine and can be used as an indicator of serotonin
production. Patients with serotonin-secreting carcinoid tumors of midgut origin usually have 5-HIAA is quantitatively
markedly elevated concentrations of urinary 5-HIAA. If there is a borderline concentration of the principal metabolite
5-HIAA in a random or 24-hour urine specimen, a repeat collection should be made with an of serotonin, and the
avoidance of foods or medications that might elevate the 5-HIAA concentrations. Only when majority of it is excreted
the 5-HIAA is normal or borderline is the measurement of serotonin needed to document the into the urine and thus
diagnosis. Platelets contain almost all the serotonin found in the blood and for that reason, the used as an indicator of
serotonin is measured in whole blood (with platelets) or in platelet-rich plasma. serotonin production.
Functioning foregut tumors may also be detected by the urinary 5-HIAA assay, even though Platelets contain almost
they secrete 5-hydroxytryptophan rather than serotonin. Urinary 5-HIAA is elevated because all the serotonin found
in the blood and for that
the 5-hydroxytryptophan released from these tumors is converted to serotonin in other tissues
reason, the serotonin is
and is subsequently metabolized to 5-HIAA. In addition, urine histamine is generally elevated in
measured in whole blood
patients with functioning foregut carcinoid tumors because these tumors (in contrast to midgut (with platelets) or in
carcinoids) usually produce histamine. platelet-rich plasma.
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Index
Page numbers in bold indicate figures and tables.
A Acute pancreatitis, 372–373 Amenorrhea

amylase, 372 laboratory evaluation of women with, 457
AAE. See Acquired angioedema
hereditary forms, 372 primary, 456
AAMPs. See Apoptosis-associated
laboratory evaluation, 372 secondary, 456–457
molecular patterns
Acute-phase reactant proteins, 10 AMI. See Acute myocardial infarction
Abacavir, 83
Acute respiratory distress syndrome Amino acidurias
ABO grouping, 298
(ARDS), 347 associated with impaired amino acid
ABO/Rh typing, 36
Acute severe anemia, nonhemorrhagic degradation, 192
Abortion, spontaneous/recurrent, 409–410
causes of, 235 clinical presentation of, 192
Accuracy, 6
Adaptive immune system, 61 diagnosis, 192
ACD. See Anemia of chronic disease
Adrenal cortex, 430 selected, 193
Acetaminophen
hypothalamic-pituitary-adrenal cortex Aminoacyl-tRNA synthetase, 68
pain management, 170–171
interactions, 431 Amylase/creatinine clearance (A/CC)
toxicity, laboratory evaluation for, 170
physiology and biochemistry, ratio, 373
Acquired angioedema (AAE), 78
430, 432 Amylase elevation
ACS. See Acute coronary syndrome (ACS)
renal and adrenal interactions, 432 renal excretion, 373
Acute bacterial infections, 109, 115
synthesis and metabolism of steroid urine, 373
Acute bacterial meningitis, 107–110
hormones, 431 Amyloid-associated protein, 71
causative organisms, 108
Adrenal medulla Amyloidosis, 70, 71
clinical presentation of, 107, 110
laboratory tests, 411–412 classification of, 71
diagnosis, 110
pheochromocytoma, 442–443 diagnosis, 72
laboratory evaluation for, 108–109
physiology and biochemistry, 411 laboratory evaluation, 73
Acute blood loss, 235–236
Adrenal sex steroids, 430 type AA amyloidosis, 71
cardinal manifestations, 235
Aerobic bacterial infections, 138, 140 Amyloid protein
diagnosis, 236
Aerobic gram-negative bacilli, 93 chemical classification, 72
nonhemorrhagic causes, 235
Aerobic gram-negative cocci, 93 Anaerobic gram-negative bacilli, 93
Acute coronary syndrome (ACS), 210, 376
Aerobic gram-negative coccobacilli, 93 Anaerobic gram-negative cocci, 93
Acute hepatocellular disease, 360
Aerobic gram-positive bacilli, 93 Analytical and statistical concepts
Acute infections, due to bacteria, 115–117
Aerobic gram-positive cocci, 93 in data analysis, 2
Acute leukemia, diagnosis of, 328
Agammaglobulinemia, 74, 75 Analytical interferences, in laboratory
Acute lymphoblastic leukemias (ALL), 320,
Age, effect on laboratory tests, 7 testing, 8
323, 331, 334
AIS. See Androgen insensitivity syndrome Anaphylactic transfusion reactions, 305
Acute myeloid leukemias, 328, 331
Alanine aminotransferase (ALT), Anaplasma (formerly Ehrlichia)
classification, 330
358–360, 364 phagocytophilum, 100
diagnostic techniques for, 331–332
Alkaline phosphatase (ALP), Androgen deficiency, 400–401
laboratory approaches for determination
358–360 Androgen insensitivity syndrome (AIS),
of prognosis, 332
Allogeneic transfusion, 310–311 453–454
prognostic markers, 332
Alpha-1 antitrypsin deficiency, 360 Androstenedione, 451
Acute myocardial infarction (AMI), 209
Alpha-fetoprotein (AFP), 400, 407 Anemia, 221
clinical history, 209–210
maternal serum, 408 acute blood loss, 235
definition, 209
Alveoli anemia of chronic disease (ACD), 237
diagnosis of, 210, 212
adjacent capillaries, thickened classification, 223
cardiac troponin, 211–216
interface, 341 definition, 221
criteria for, 211
impaired ventilation, airway diseases, diagnostic algorithm
cutoff for, 214
340–341 macrocytic, 224
prevalence, 209
Alzheimer amyloid precursor protein microcytic, 223
Acute normovolemic hemodilution
(APP), 71 normocytic, 224
(ANH), 310
Amblyomma americanum, 100 suspected hemolytic, 225
469
470 INDEX
Anemia (Cont’d.) APP. See Alzheimer amyloid Biomarkers

differential diagnosis, 222 precursor protein of acute kidney injury, 396
abnormal morphologies/intracellular ARDS. See Acute respiratory CA 15-3, 416
inclusions of RBCs, 227–235 distress syndrome CA 27.29, 416
morphologic findings in red cells, 226 Arthralgias, 69 carcinoembryonic antigen (CEA), 416–417
folate deficiency, 239 Arthritis, 73 estrogen receptor (ER), 414
hemolytic, 235 Aspartate aminotransferase (AST), HER2, 414–415
intravascular/extravascular 358–360, 364 progesterone receptor (PR), 414
hemolysis, 235 Aspirin, 162, 177, 260, 288 serum-based, 416–417
iron deficiency, 236 pharmacokinetics of, 171–172 tissue-based, 414–415
diagnosis, 236–237 toxicity, laboratory evaluation for, 171 Biphenotypic leukemias, 331
free erythrocyte protoporphyrin Atherosclerosis, 198 Blackfan-Diamond syndrome, 222, 248
(FEP), 237 causes of, 198 Bleeding disorders, 259
lead poisoning, 237 diagnosis, 198–202 antiplasmin/plasmin inhibitor, 267
microangiopathic hemolytic, 245 major plasma lipoproteins, 199 Bernard–Soulier syndrome (BS), 278
sickle cell anemia, 241 Atherosclerotic vascular disease, 2, 197–199, coagulation factor, 259–260, 263
stages of, 236 198, 201 contact factors, deficiencies of, 267
thalassemia, 238 Autoimmune diseases, 62, 64, 69, 70, 75, 77, disseminated intravascular coagulation, 269
vitamin B12 deficiency, 239 78, 120, 354–355, 424, 428 drug-induced immunologic
WHO definition, 222 Autoimmune disorders, 63, 76, 319, 425 thrombocytopenia, 272–273
zinc protoporphyrin (ZPP), 237 involving connective tissue drug-induced qualitative platelet
sideroblastic, 247 approach to diagnosis, 63 disorders, 280–281
Anemia of chronic disease (ACD), 237 Autoimmune hemolytic anemia, 243–244 essential thrombocythemia, 275
definition, 237 Autoimmune myositis, 69 Factor V deficiency, 262
diagnosis, 237 Automation, 14 Factor VII deficiency, 262
Angiotensin-converting enzyme Autosomal dominant disorder, 243, 363, 417 Factor X deficiency, 259, 265
inhibitors, 392 Factor XI deficiency, 266
Angiotensin II receptor blockers, 392 Factor XIII deficiency, 267
ANH. See Acute normovolemic B fibrinogen deficiencies, 259–261
hemodilution (ANH) Babesiosis, 102 fibrinolytic pathway factor deficiencies,
Anion gap, calculation of, 343, 346 causative pathogens, 102–103 259–260
Anti-basement membrane diagnosis, 103 Glanzmann thrombasthenia (GT), 254,
autoantibodies, 386 laboratory evaluation, 103 260, 278, 279
Antibody screen, 299 Bacillary angiomatosis, 120 hemophilia A, 264
Anticardiolipin antibodies, 284 Bacillus cereus, 145 factor VIII procoagulant activity, 264
Anticoagulant therapies, 287 Bacteremia, 98 hemophilia B, 265
Antigen detection, 97 causes of, 98 factor IX procoagulant activity, 265
direct and indirect immunofluorescence diagnostic evaluation, 98–99 hemostatic defects, 271, 280
for, 25 Bacteriuria, 394 uremia, 280
Antiglobulin technique crossmatch, 301 Balantidium coli, 141 idiopathic/immune thrombocytopenic
Antihistidyl-tRNA synthetase, 68 Barbiturates, 163, 172, 176, 363 purpura (ITP), 271–272
Anti-HPA-1a antibody, 273, 274 BC. See Bilirubin, conjugated liver disease, 270–271
Antihuman globulin (AHG), 87, 251 B cells, 61, 75, 77 hemostatic abnormalities, 270
Anti-Jo-1, 68 neoplasms, 321 neonatal alloimmune thrombocytopenia
Anti-Mi-2 antibodies, 68 Benign prostatic hyperplasia (BPH), 398 (NAIT), 274–275
Antimicrobial sensitivity tests, 24 Bentiromide test, 374 platelet storage pool disease (SPD),
Antineutrophil cytoplasmic antibody Benzodiazepines, 172, 173, 175, 176 278–280
(ANCA) testing, 198 Bernard-Soulier disease, 278 posttransfusion purpura (PTP), 273–274
Antinuclear antibody (ANA) testing, 15 laboratory evaluation, 279 prothrombin (Factor II) deficiency, 262
diseases associated with positive test Bernard-Soulier syndrome (BS), 278 vitamin K deficiency, 268
results for, 62 Beta cell tumors, 375, 382 von Willebrand disease (vWD), 275–278
scleroderma, 67 Beta-2 glycoprotein I antibodies, 284 Bleeding time, 14
Antiphospholipid antibodies, 206, 284, 285 Beta-hemolytic streptococci, 98 Blood cells
Antiplasmin deficiency, 267–268, 268 Beta-2 microglobulin, 326 counting with automated white blood
laboratory evaluation, 268 Biliary tract disease, 358, 359, 369 cell differential count, 26
Antiplatelet therapies, 288 ALT/AST/LDH/ALP, 359–360 Blood collection, 292
Anti-signal recognition particle (SRP) causes of, 361 cap color and contents of tubes for, 10
antibodies, 68 Biliary tract obstruction, 363 patient posture for, 8
Antisynthetase antibodies, 68 Bilirubin, 190, 225, 360–364, 395 timing of, 9–10
Antisynthetase syndrome, 68 bilirubinuria, 361 Blood component
Apheresis, 41 conjugated (BC), 361, 363 derivatives, and indications for use, 295
Apolipoprotein A-1 gene defects, 202 hyperbilirubinemia, 362, 363 descriptions and indications, 294–295
Apoptosis-associated molecular patterns measurement, 362 preparation, 37
(AAMPs), 61 unconjugated, 361, 362 Blood component preparation, 37, 293
INDEX 471
Blood crossmatch, 38 primary treatment, 413 encephalitis, 111–112

Blood cultures, 23 risk of developing, 413 sites of, 107
clinical significance of organisms, Breast cancer syndrome, 417–418 CGD. See Chronic granulomatous disease
isolated from, 100 BRCA1/BRCA2 mutations, 417 CGH. See Comparative genomic
Blood donors, 73, 292, 296 hereditary, 417 hybridization
Blood gases, 342 Bronchoalveolar lavage fluid analysis, 345 Chediak-Higashi syndrome, 337
functional abnormalities btk gene, 75 Chemotherapy, 92, 127, 259, 311, 314,
detected with battery of tests, 342 BUN. See Blood urea nitrogen 331, 453
measurements, 45 BUN/Creatinine (Cr) ratio, clinical use of, 390 adjuvant, 413, 415
selected disorders associated Buprenorphine, 161, 162, 177 cell death from, 396
with acid–base abnormalities, 343 Burkitt lymphoma, 104 leukemogenic, 328
with hypoxemia, 342 malignant transformation, 410
test panel, 342 Childhood disorders, 186
Blood gas test panel, 342 C Chlamydial infections, 152–153
Blood glucose values CA 19-9 antigen, 375 causative organism, 152
differences in test results, 8 CAIHA. See Cold autoimmune hemolytic diagnosis, 153
Blood parasites, 102 anemia diseases caused by, 152–153
Blood pH, 342 Calcitonin, 425, 430, 444, 464 evaluation of patient for, 154
Blood transfusion, complications of, 191, Calcium, 396 Chlamydia trachomatis, 149
235, 304 Canalicular multispecific organic anion Chlamydophila pneumoniae, 152
immunologic reactions, 304–307 transporter (cMOAT), 363 Cholangitis, 145, 361, 363, 369
platelet reactions, 306–307 Candida albicans, 148 Cholesterol ester transfer protein (CETP)
RBC reactions, 304–305 Candidemia, 101 deficiency, 202
reactions to plasma components, 305 Cannabinoids, 173, 175, 176 Cholesterol level, 7
white blood cell reactions, 306 Carbamazepine, 161, 166, 168 Choriocarcinoma, 400, 410
nonimmunologic reactions, 307–309 Carbon monoxide poisoning, 179 Chromatography
chemical complications, 308 clinical presentation, 180 for separation, identification, and
complications created by physical diagnosis of, 180–181 quantitation of, 50
characteristics of blood, 307 laboratory monitoring, 180 Chromomycosis, 121, 122
infectious complications, 308–309 pathologic consequence of, 180 Chronic granulomatous disease
transfusion reaction workup, 309–310 Cardiovascular risk, assessment of, 201 (CGD), 337
Blood urea nitrogen (BUN), 386, 389–390 Framingham score, 201 Chronic hyperglycemia, 376
albumin excretion, 391 PROCAM score, 201 Chronic hypocalcemia, 448
to creatinine ratio (BUN/Cr), 390 Reynolds score, 201 Chronic lymphocytic leukemia (CLL),
modern laboratory methods, 389 systematic coronary risk evaluation 244, 320, 323
nitrogen retention, 386 (SCORE), 201 Chronic meningitis, 101, 107, 111
BMT. See Bone marrow transplantation Cat-scratch disease, 120 Chronic myeloid leukemia, 333
Body mass CBAVD. See Congenital bilateral absence of characterized by, 333
effect on laboratory tests, 8 the vas deferens diagnosis, 334
Bone formation markers, 446 Celiac disease, 62 stage of disease, for patients, 334
alkaline phosphatase (ALP), 446–447 Celiac sprue treatment, 334
procollagen type I intact N-terminal biopsy, 354 Chronic myeloid leukemia, 329, 333, 336
propeptide (PINP), 447 diagnostic tests, 354 Chronic myelomonocytic leukemia
serum osteocalcin, 447 deamidated gliadin antibodies, 353 (CMML), 329, 336–337
Bone infections, 112–113 gliadin antibodies, 353 Chronic obstructive pulmonary disease
Bone infections/osteomyelitis, 112 small bowel biopsy, 354 (COPD), 339, 340, 343, 345, 346
Bone marrow biopsy, 73, 74, 249, 318, 336 tissue transglutaminase (TTG) IgA Chronic pancreatitis, 374–375
Bone marrow transplantation (BMT), 77, antibodies, 354 laboratory evaluation, 372
83, 87, 105, 136, 333, 334 immunologic disease, 353 Chronic renal failure, 217, 280, 386, 461
Bone mineral density (BMD), 450 nutrients malabsorption, 353 Chymotrypsin, 374
Borrelia burgdorferi, 114, 118, 119 serologic tests, 353–354 cleaves, p-aminobenzoic acid (PABA)
Botulism, 145–146 symptoms, 353 molecule, 374
laboratory evaluation, 146 Cell injury CID. See Cellular immunodeficiency
BPH. See Benign prostatic hyperplasia and effects inflammation on selected diseases
Brain abscess, 112 laboratory tests, 10 Cirrhosis, 360
Breast cancer, 413 Cellular immunodeficiency diseases Citrate toxicity, 308
adjuvant therapy, 414 (CID), 77 Clinical laboratory methods, 249
high-penetrance cancer predisposition Cellular therapies, 311–312 direct antiglobulin test (Coombs test), 251
genes, 418 Central nervous system, infections, 107 hemoglobin electrophoresis, 250
Cowden syndrome, 418 acute bacterial meningitis, 107, 110 Kleihauer–Betke (acid elution) test, 251
Li-Fraumeni syndrome, 418 acute viral meningitis, 110 osmotic fragility test, 251
Peutz-Jeghers syndrome, 419 brain abscess, 112 red cell indices, 249–250
laboratory testing, 414–417 chronic meningitis, 111 reticulocyte counting, 250
Mortality, 413 diagnosis, 110 screening tests for sickle hemoglobin, 251
472 INDEX
CLL. See Chronic lymphocytic leukemia Component preparation, 293 Data analysis, 2
Clostridial infections, 138 Congenital bilateral absence of the vas Deep vein thrombosis (DVT), 197, 198
Clostridium difficile infections, 138 deferens (CBAVD), 402 congenital risk factors for, 206
Clot formation, 254 Congenital T-cell immunodeficiencies, 306 diagnosis, 206–207, 207
fibrin clot formation, 255–258 Congestive heart failure, 216 incidence, 206
fibrinolysis, 258–259 biological variability, 218 Dermacentor variabilis, 100
platelet plug formation, 254–255 clinical presentation of, 216 Dermatomyositis, 67, 68
CMML. See Chronic myelomonocytic diagnosis of characteristic feature, 68
leukemia implications for, 218 fulfill clinical diagnostic criteria, 68
cMOAT. See Canalicular multispecific medical decision cutoff values, 218 Desirable range, 2
organic anion transporter natriuretic peptide measurement, Diabetes mellitus (DM), 371, 376–378
CMV. See Cytomegalovirus 216–217 ADA classification system, 379
Coagulation factors, 7 myocardial infarction (See Myocardial American Diabetes Association
assays, 33 infarction) Classification, 377
deficiencies, 259, 262 Conn syndrome, 439 blood glucose levels, 378
laboratory evaluation, 263 Contact coagulation factors, 267 criteria for diagnosis, 376
disorders, 260 Contact factors, deficiencies, 267 fasting blood glucose, 8, 378
Coagulopathies, 73, 254, 269, 281 Coombs test, 243, 301, 362 gestational diabetes, 380–381
Cocaine, 172, 173, 176–177 Coumadin therapy. See Warfarin therapy high-risk, 377
Coccidia, 94–95, 140 Counting of blood cells, 26 noninsulin-dependent, 377
Coccidioides spp., 135, 136 Cowden syndrome, 418 oral glucose tolerance test
Coccidioidomycosis, 135 C-reactive protein (CRP), 10, 72 (OGTT), 378
Cold autoimmune hemolytic anemia Creatine kinase (CK), 69, 117, 210, prediabetes, 377
(CAIHA), 243, 313 359, 387 random blood glucose, 378–379
Collection of blood Creatinine clearance (CC), 373 urine glucose, 394
and preparation of blood CREST syndrome, 67 Diabetic nephropathy, 391
components, 292 Creutzfeldt-Jakob disease, 72, 94, 309 blood pressure findings, 392
Colonoscopy, 354–355 CRH stimulation test, 433 Diagnostic cutoff, need for, 3, 439
Colorectal cancer, 354–356 Crigler–Najjar syndrome type II, 363 Diagnostic threshold values
genetic profiling, 355–356 Cryoglobulin identification of the appropriate value, 4
screening test, 355 analysis, 20 to minimize number of false reactions, 5
colonoscopy, 355 types, 73 DIC-like syndrome, 270
fecal immunochemical testing Cryoglobulinemia, 70, 73 Dientamoeba fragilis, 140
(FIT), 355 diagnosis, 74 DiGeorge syndrome, 77, 306
fecal occult blood testing laboratory evaluation, 74 causes, 77
(FOBT), 355 Cryoprecipitate, 304 diagnosis, 77
Common variable immunodeficiency Cryptococcus neoformans, 101, 102 thymic dysfunction, 77
(CVID), 75 Cultures, 96 Digoxin, 161, 163, 166, 170
laboratory evaluation for, 76 identification of organisms from positive Dihydroxyphenylalanine (DOPA), 441
Comparative genomic hybridization cultures, 96 Dilute Russell viper venom time (DRVVT)
(CGH), 57 Cushing syndrome, 204, 343, assays, 284
Compatibility testing, 297 433–436, 457 Dimorphic fungi infections, 111, 135–136
ABO grouping, 298–299 CVID. See Common variable 2,3-Diphosphoglycerate, depletion of, 308
antibody screen, 299–301 immunodeficiency Direct antiglobulin test (DAT),
direct antiglobulin test, 301–302 Cyclosporine, 166 39, 251, 301
direct Coombs’ test, 301–302 Cystatin C, 395 Direct stains, 96
indirect antiglobulin assay, 299–301 Cystic fibrosis Disorders associated with impaired WBC
for other blood components that do not clinical presentation of, 191 function, 337
contain RBCs, 302 laboratory evaluation for, 192 Disseminated intravascular coagulation,
pretransfusion testing, 297–298 screening and diagnosis, 192 269–270
RBC crossmatch, 301 Cytapheresis, 314 laboratory evaluation, 270
Rh system, 299 Cytomegalovirus (CMV), 103, 104 DM. See Dermatomyositis; Diabetes mellitus
Complement activation, patterns, 79 clinical syndromes, 104 DNA-based diagnostic tests, 186
Complement proteins deficiencies congenital infections, 105 DOPA. See Dihydroxyphenylalanine
acquired C1 INH, 78 diagnosis, 105 Down syndrome, 407
C5, C6, C7, and C8 result in, 77–78 laboratory testing, 105 causes of, 188
complement activation, useful in clinical characteristics, 188
diagnosis, 79 laboratory evaluation for, 189
C1q, C2, and C4 predispose to, 78 D screening and diagnosis, 188–189
decay accelerating factor, 78 DAMPs. See Danger-associated Drug-induced immunologic
diagnosis, 78–79 molecular patterns thrombocytopenia, 272–273
factor I and H, 78 Danger-associated molecular patterns clinical causes, 272
homologous restriction factor, 78 (DAMPs), 61 diagnosis, 273
inherited, evaluation for, 79 DAT. See Direct antiglobulin test laboratory evaluation, 273
INDEX 473
Drug-induced qualitative platelet Environmental toxins, 179 Female genital system, 405–412
dysfunction, 280, 281 carbon monoxide, 179 abortion
diagnosis, 281 clinical presentation, 180 laboratory evaluation, 410
Drug of abuse testing (DAT), 172–174 laboratory monitoring, 180 spontaneous/recurrent, 409–410
characterization of, 174 lead, 181 gestational trophoblastic disease, 410
considerations in, 160, 166 diagnosis, 181 HELLP syndrome, 411
false-positive, 173–174 laboratory monitoring, 182 infertility, 411–412
goal of, 160, 172 Enzyme-linked immunosorbent assay preeclampsia/eclampsia, 410–411
Drugs impact, on laboratory test results, 8 (ELISA), 47, 67 pregnancy
Drugs of abuse, 172 Eosinophils, 319, 327, 329 abnormal pregnancy conditions, 409
characteristics of immunoassay tests EPO. See Erythropoietin ectopic, 408–409
for, 173 Epstein–Barr virus (EBV), 103, 104 effects on select laboratory tests, 406
“confirmatory” testing, 174 associated with, 104 fatty liver of, 411
detection, 3 diagnosis, 104 normal, 405–407, 406
window for commonly abused ERCP. See Endoscopic retrograde screening
substances, 172 cholangiopancreatography maternal serum, 407–408
selected drugs, 175 Errors minimization sequential serum, 407
alcohols, 177–179, 178, 179 in interpretation of laboratory test Female genital tract, 148
amphetamines, 175 results, 7 diagnosis, 148
barbiturates, 176 in selection of laboratory tests, 7 infections, 148, 149
benzodiazepines, 176 Erythroblastosis fetalis. See Hemolytic Fentanyl, 163, 172, 177
cannabinoids, 176 disease of the newborn (HDN) Fetal lung maturity assessment
cocaine, 176–177 Erythrocytapheresis, 314 laboratory tests, 347
opiates and opioids, 177 Erythrocyte sedimentation rate (ESR), FFP. See Fresh frozen plasma
phencyclidine, 177 10, 30 Fibrinogen deficiencies, 259
specimen collection and laboratory Erythrocytosis, 248–249 laboratory evaluation, 261
analysis, 174–175 definition, 248 Fibrinolysis, 258–259
Dry eyes (keratoconjunctivitis), 65 differential diagnosis, 248 FISH. See Fluorescence in situ hybridization
Dry mouth (xerostomia), 65 polycythemia vera, 248 FIT. See Fecal immunochemical testing
Dubin–Johnson syndrome, 363 criteria for, 249 Flow cytometry, 18
DVT. See Deep vein thrombosis Erythropoietin (EPO), 222, 223, 249, 310, Fluorescence in situ hybridization (FISH),
Dyspepsia, 351, 352 385, 386, 387, 393 77, 323, 325, 331, 334, 415
ESR. See Erythrocyte sedimentation rate FOBT. See Fecal occult blood testing
Essential thrombocythemia, 275, 335 Folate deficiency, 239
E Estradiol, 7, 405, 454, 455, 456 causes, 239
Echinococcus granulosis, 141 Estriol, 405, 408 diagnosis, 239
Eclampsia, 410–411 Estrogens, 455 dietary factors, 239
Ectopic pregnancy, 408 Estrone, 405 Follicle-stimulating hormone (FSH), 407
Ehrlichia chafeensis, 100 Ethanol abuse, 177–179, 179 estrogen and progesterone levels, 400
Electrolyte measurements laboratory evaluation, 178 inhibin A, 408
chloride, 43 Euthyroid sick syndrome (ETS), 426, 429 midluteal progesterone
potassium, 43 Extravascular hemolysis, 225, 227, 235, 244 concentrations, 412
sodium, 43 Eye infections, 124 Food ingestion, 178, 379, 445
Electrolytes causative organisms, 124–125 Food poisoning, 139–140, 145
definition, 342 and causative organisms, 124–125 Fractional excretion of sodium (FENa), 392
measurements of, 43, 343 diagnosis, 124 as an indicator of tubular function,
Electrophoresis 392–393
hemoglobin, 29, 241, 242 Fresh frozen plasma (FFP), 37, 293, 294,
lipoprotein, 14 F 303, 304
serum and urine protein, 73, 392, 393 FAB system, 328 Fully automated immunoassay
Encephalitis, 111 Factor V deficiency, 262 with all reagents in solution, 48
laboratory evaluation, 108–109 Factor VII deficiency 262 Fungal infections, 120
Endocarditis, 106, 107, 115 Factor VIII Inhibitors, 264–265 evaluation, 122
Endocrinologic disorders, 455–457 laboratory evaluation, 265 Fungemia, 101, 113, 125
affecting female reproduction, 455–457 Factor X deficiency, 266 causes of, 101
changes hormones in menstruating Factor XI deficiency, 266–267 diagnosis, 101–102
females, 456 Factor XIII deficiency, 267 Fungi, of medical significance, 93
approach to patient with, 422 Familial combined hyperlipidemia, 202
Endolimax nana, 140 Familial HDL deficiency, 203
Endoscopic retrograde Familial hyperchylomicronemia, 202 G
cholangiopancreatography Fasting plasma glucose (FPG) test, 378 Gaisbock syndrome, 248
(ERCP), 374, 375 Fatigue, 69 Gamma-glutamyltransferase, 359
Entamoeba histolytica, 140, 141 Fecal immunochemical testing (FIT), 355 Gamma-glutamyl transpeptidase (GGT),
Enterocytozoon bienusi, 141 Fecal occult blood testing (FOBT), 355 178, 358, 359, 370
474 INDEX
Gardnerella vaginalis, 149 Granulocyte–monocyte colony-stimulating Hepatic glutathione-S-transferase levels

Gastrinomas, 375 factor (GM-CSF), 89 (hGSTA1-1), 363
Gastroesophageal reflux disease Granulocytopenic disorder, Hepatitis A virus (HAV), 365
(GERD), 351 classification, 318 Hepatitis B virus (HBV), 365
Gastrointestinal tract, infections Granulomatous prostatitis, 148 Hepatitis C virus (HCV), 365
aerobic bacterial infections, 138, 140 Grave’s disease, 424–427 Hepatitis D virus (HDV), 365
botulism, 145–146 Gray platelet syndrome, 279 Hepatobiliary system
clostridial infections, 138 Growth hormone excess/deficiency detoxifying functions, 361–364
evaluation for viral, 138 laboratory evaluation, 459 excretory functions, 361–364
food poisoning, 145 Growth hormone (GH) secretion, Hepatocellular disease, causes of, 360
helminth infections, 141, 143 regulation of, 458 Hereditary elliptocytosis (HE), 243
protozoal infections, 140–141 GSD. See Glycogen storage disease Hereditary spherocytosis, 242–243
viruses inducing gastroenteritis, 137–138 GT. See Glanzmann thrombasthenia Herpes simplex, 149
Gaucher disease, 360 Herpes simplex virus infections, 153–155
GDM. See Gestational diabetes mellitus evaluation for, 155
Gender, effect on laboratory tests, 7 H laboratory diagnosis, 154
Genital system. See Female genital system Haemophilus influenzae, 74 transmission, 153–154
GERD. See Gastroesophageal reflux disease Hairy cell leukemia, 324 type 1 and type 2, 153
Germ cell tumors, 399, 400 Haptoglobin, 191, 225, 235, 364, 370, 393 High-sensitivity C-reactive protein,
Gestational choriocarcinoma, 410 Hashimoto thyroiditis, 62 200–201
Gestational diabetes mellitus (GDM), HDL-associated abnormalities, 202 Histidyl-tRNA synthetase, 68
380–381 HDL cholesterol, 199, 200 Histocompatibility antigens, 83
ADA criteria, 380 HDN. See Hemolytic disease of the Histocompatibility testing assays, 86
laboratory evaluation, 381 newborn crossmatching, 87
screening for, 380–381 HE. See Hereditary elliptocytosis HLA antibody screening, 86
Gestational trophoblastic diseases, 410 Heat shock proteins (HSP72), 396 HLA typing, 86
GFR. See Glomerular filtration rate Heavy chain disease, 326 Histoplasma capsulatum, 101, 102
GGT. See Gamma-glutamyl transpeptidase Helicobacter pylori infection, 375 HIV infection, 92
Giardia lamblia, 140 HELLP syndrome, 411 HLA allele, 85
Giardiasis, 75 Helminth (worm) infections, 141, 143 HLA and ABO blood group matching in
Gilbert syndrome, 363, 369 diagnosis, 141 transplants, 87
Glanzmann thrombasthenia (GT), 254, Hematopoietic stem cell transplantation, HLA-B27, in ankylosing spondylitis, 83
260, 278, 279 88–89 HLA DR 52, 68
laboratory evaluation, 279 Hematuria, 394 HLA genes
Glasgow criteria, 373 Hemoglobin codominant expression, 85
Glomerular filtration rate (GFR), electrophoresis, 29, 241, 242, 250 and gene products, 84–85
387–390, 396 Hemoglobin A1c (HbA1c), 378, 379 serologic specifications, 85
creatine, 388 Hemoglobinopathies, 186, 222, 241, HLA nomenclature, 85
formula for clearance, 388 241–242, 250, 362 HLA typing, 86, 87
estimate of GFR (eGFR), 388 Hemolytic anemia, 235 for BMT, 87, 89
National Kidney Foundation (NKF) intravascular/extravascular for cornea transplantation, 88
advises, 388, 389 hemolysis, 235 for heart transplantation, 88
kidney damage, 389 Hemolytic disease of the newborn (HDN), for liver transplantation, 88
Glomerulonephridites, 386 190–191, 245 for lung transplantation, 88
Glomerulus, 373, 386–391 causes of, 190 for pancreas transplantation, 88
Glucagonomas, 376 laboratory evaluation, 191 for renal transplantation, 87, 88
Glucocorticoids, 430, 433, 436, 437, screening and diagnosis, 190–191 using microlymphocytotoxicity assay, 87
439, 440 Hemolytic-uremic syndrome, 286 Hodgkin lymphoma, 327
Glucose-6-phosphate dehydrogenase Hemophilia A (factor VIII deficiency), 264 Homovanillic acid (HVA), 195
(G6PD) deficiency, 225, 246, 362 Hemophilia B (factor IX deficiency), H. pylori
Glutamic acid decarboxylase (GAD65), 379 265–266 laboratory tests, 352, 352–353
Gluten proteins, 353 Hemostasis, 254 peptic ulcer disease, 352
Glycogen storage disease (GSD), 360, 366 clot formation, 254 Human chorionic gonadotropin (hCG),
Glycohemoglobin, 379 fibrin clot formation, 255–258 400, 405
measurement of, 379 platelet plug formation, 254–255 blood and urine, 407
Glycosylphosphatidylinositol (GPI), 247 fibrinolysis, 258–259 dilation and evacuation (D&E)
Gonorrhea, 150, 152 fibrinolytic pathway, 258 procedures, 410
causative organism, 150 plasmin, 258 ectopic pregnancies, 409
diagnosis, 152 plasminogen predisposes, deficiency Human MHC system, genes of, 84
sample collection site, 152 of, 258 Hydatidiform moles, 410
Goodpasture syndrome, 313, 345, 386 Hemostatic abnormalities in liver disease, 5-Hydroxytryptamine, 465
Gram stain, 11, 21 270–271 Hyperaldosteronism, 438
Granulocyte colony-stimulating factor laboratory evaluation, 271 Hypercalcemia of malignancy, 449–450
(G-CSF), 89 Hemostatic defects, in uremia, 280 Hypercoagulable states, 281
INDEX 475
Hyperglycemia, 201, 372, 376, 377, 379, Indirect antiglobulin test (IAT), 40, 299 International normalized ratio (INR),
380, 391, 463 Infections 2, 287, 288, 303, 304, 365
nonketotic, 193 aerobic bacterial, 138 Interpretations
Hyper-IgM syndrome (HIGM), 76 bacterial, acute, 115 of clinical laboratory test results, do not
diagnosis, 76 of blood, viral, 103 involve range, 3
Hyperparathyroidism, 387, 444 caused by Rickettsia, Ehrlichia, and ranges used in test results, 2
primary, 447–448 related organisms, 100 Intravascular hemolysis, 225, 305, 394
secondary, 448 of central nervous system, 107 Iron deficiency, 223, 224
Hypertension, 203 chlamydial, 152 Islet cell tumors, 375–376, 382
causes of, 203 Clostridium difficile, 138 Islet tyrosine phosphatase (ICA512), 379
diagnosis, 204 of female genital tract, 148 ITP. See Immune thrombocytopenic purpura
evaluation of patient for correctable of gastrointestinal tract, 137
causes of, 204 general clinical approach to the patient
Hypertensive vascular disease, 198 with, 95 J
Hypoaldosteronism, 438 of heart (See Infectious endocarditis) JAK2 protein, 333
Hypocalciuric hypercalcemia, 450 herpes simplex virus, 153 Janus kinase 2 (JAK-2) mutation, 249
Hypocomplementemia, 73 intestinal helminth, 141 Jaundice, 144, 242, 285, 360, 363, 369
Hypogammaglobulinemia, 75 of joints, 113 Joint infection
Hypoglycemia, 381–382 of larynx, pharynx, mouth, ear, orbit, and clinical presentation, 113–114
Hypoglycemic patients sinuses, 125, 126–127 diagnosis, 114
fasting, 381 Legionella, 131 evaluation for organisms associated
plasma glucose level in, 381 of lung and pleurae, 127, 130 (See also with, 114
reactive, 381 Lung infections)
sulfonylureas, excess administration, 382 of male genital tract, 148
surreptitious insulin injection, 382 parasitic (See Parasitic infections of blood) K
Hypogonadism, 454 in perinatal period, 189 Karyotype, evaluation of chromosomes, 55
Hypoparathyroidism, 444, 448–449 screening and diagnosis, 190 Kayser-Fleischer ring, 367
laboratory evaluation, 448 protozoal infections, 140 Ketones, 395
Hypotension, 98, 139, 305, 307, 440, 441 pyelonephritis and urinary tract, 146 17-ketosteroids, 451, 453
Hypothalamic-pituitary-adrenal cortex respiratory virus, 137 Kidney. See also Renal disease
interactions, 431 sepsis and bloodstream, 98 damage, 389
Hypothalamic-pituitary-gonadal axis, 452 of skin and adjacent soft tissue, 115 homeostatic roles, 385
Hypothalamic-pituitary-thyroid testing, 296 nephrolithiasis/urolithiasis, 395
interactions, 395 of donated blood, 297 urinary casts, 394
Hypothyroidism, 186, 203, 427, 428, 430, varicella zoster, 121 vitamin D, 387
456, 457 Infectious diseases. See Infections Kleihauer–Betke test, 251
Hypoxemia disorders, 342, 347 Infectious endocarditis, 106
clinical features of, 106
diagnosis, 106 L
I evaluation of patient with, 107 Laboratory performance
IFE. See Immunofixation electrophoresis risk factors for, 106 analyzing errors, 6–7
IFG. See Impaired fasting glucose Infectious mononucleosis, 104 Laboratory tests
IGT. See Impaired glucose tolerance evaluation for, 104 effect of age on, 7
IHC. See Immunohistochemistry Infertility, 411 effect of body mass on, 8
IMGT/HLA Database, 85 female, 411–412 effect of gender on, 7
Immune thrombocytopenic purpura (ITP), male, 401–402 for infectious agents, 96
271–272 Inflammation antigen detection, 97
laboratory evaluation, 272 and effects of cell injury on selected culture, 96
Immunodeficiency diseases, 62 laboratory tests, 10 direct stains, 96
primary, 62 markers of, 10 identification of organisms from
secondary, 62 Inflammatory muscle diseases, 66, 67–69 positive cultures, 96–97
Immunoelectrophoresis, 14 dermatomyositis (DM), 67 nucleic acid amplification, 97
Immunofixation electrophoresis (IFE), 392 inclusion body myositis, 67 serology, 98
Immunofixation to identify monoclonal laboratory evaluation, 69 susceptibility testing, 97
immunoglobulins, 17 polymyositis (PM), 67 results
Immunofluorescence for antigen INR. See International normalized ratio impact of drugs, 8
detection, 25 Insulin-like growth factors (IGFs), 458, 459 preanalytical variables affecting, 7
Immunohistochemistry (IHC), 129, Insulinomas, 375, 381–382 used in evaluation of liver function, 366
322–323, 414 Intact hormone (iPTH), 446 acute (fulminant) hepatic failure,
Immunologic reactions, 304 Interferences 368–369
Impaired fasting glucose (IFG), 377, 378, 381 in laboratory tests, 8 alpha-1 antitrypsin deficiency, 366
Impaired glucose tolerance (IGT), 374, 377, analytical interferences, 8 alpha-fetoprotein (AFP), 367
378, 381 impact of drugs, 8 cholestasis of pregnancy and serum
Inclusion body myositis, 67 longitudinal analysis, 3 bile acid, 368
476 INDEX
Laboratory tests (Cont’d.) alpha-1 antitrypsin deficiency, 360, 366 Malaria, 102
cirrhosis, 369 alpha-fetoprotein (AFP), 367 causative pathogens, 102
glycogen storage diseases, 366 ammonia, 368 diagnosis, 102
hemochromatosis, 367 cirrhosis, 360, 364, 369 laboratory evaluation, 103
hepatic encephalopathy and glycogen storage diseases, 360, 366 Male genital tract, 397–403
ammonia, 368 hemochromatosis, 367 diagnosis, 148
hepatocellular carcinoma, 367 hepatic encephalopathy, 368 infections, 148
primary biliary cirrhosis, 369 hepatocellular carcinoma, 367 Male gonadal dysfunction, 400–401
primary sclerosing cholangitis, 369 pregnancy, cholestasis of causes of, 400
Wilson disease, 367 primary biliary cirrhosis, 369 diagnosis, 401
Lactate dehydrogenase (LD), 359, 400 primary sclerosing cholangitis, 369 Male infertility, 401–403
activity, 411 serum bile acid concentrations, 368 causes of, 401–402
red blood cells, 400 Wilson disease, 367 absence of spermatozoa, 402
Lamellar body counts (LBC), 347 patient, approach, 369–370, 370 vasa deferentia, 402
Lamotrigine, 161, 166, 169 synthetic function of, 364 diagnosis and laboratory test
Latent tuberculosis infection (LTBI), 130 viral hepatitis test, 365, 366 interpretation, 403
Latex agglutination, 49 vitamin K malabsorption, 364, 365 Markers
Latex agglutination, 49 Liver enzyme, 358–359, 363, 369 of inflammation and acute-phase
LD. See Lactate dehydrogenase (LD) Liver function evaluation, 366 response, 10
LDL cholesterol, 199 Liver parenchyma, 369 release of plasma markers of organ
associated abnormalities, 202 Liver plasma membrane integrity, enzymes damage from injured cells, 10
direct measurement of, 199, 200 indicative of, 358 Massively parallel genomic sequencing
Lead poisoning, 181, 226, 240, 241 Liver, urea, 389 (MPGS), 408
definition, 240 LTBI. See Latent tuberculosis infection Mass spectrometry
diagnosis, 181, 241 Lung cancer, 339 for molecular identification, 51
laboratory monitoring, 182, 241 Lung disorders, 346 Maternal serum screening, 407–408
renal toxicity, 181, 240 asthma, 346 MCTD. See Mixed connective tissue disease
Lecithin, 188 chronic obstructive pulmonary MDRD. See Modification of diet in renal
Lecithin cholesterol acyltransferase (LCAT) disease, 346 disease
deficiency, 202 lung cancer, 339, 348 Measles. See Rubella; Rubeola
Legionella infections, 131 respiratory distress syndrome, 347 Meningitis
diagnosis, 133 Lung infections chronic, 111
subclinical infections, 133 by dimorphic fungi, 134–135 laboratory evaluation, 108–109
Legionella pneumophila, 131 legionellosis, 132–133 Metabolic disorder, 187
Legionellosis, 133 nocardiosis, 133–134, 135 inherited, routine laboratory screening
Leukemias with recurrent genetic Pneumocystis jirovecii pneumonia for, 187
abnormalities, 328–329 tuberculosis, 130–131 Metabolic syndrome, 201, 367
Leukemogenic function, 329 Lupus anticoagulant, 284 Metanephrine, 441–443
Leukocytes, 104, 105, 139, 307, 312, Luteinizing hormone (LH), 407 Methods. See Clinical laboratory methods
314, 403 Lyme disease, 102, 118 Methotrexate, 161, 166, 167, 409
Leukopenia, 318 Lymphocytes, 319 N-methylthiotetrazole (MTT), 268
accelerated removal of granulocytes, Lymphoid malignancies MHC. See Major histocompatibility complex
caused by, 319 B/T-cell neoplasms, 320, 323 Microalbuminuria, 380
defects in production of granulocytes, chronic lymphocytic leukemia, 323–324 interpretation of albumin excretion, 391
caused by, 318–319 classification of, 320, 321 Microangiopathic hemolytic anemias,
diagnosis, 318–319 hairy cell leukemia, 324 245–246
immunodeficiency diseases associated heavy chain disease, 326–327 Microbiologic culture, and organism
with, 318 hodgkin lymphoma, 327–328 identification, 22
Leukopenia, 100, 318–319, 324 lymphoma, 322–323 Microorganisms, clinically significant,
Levetiracetam, 161, 161, 166, 169 monoclonal gammopathies, 327 93–95
Lewis blood group antigen, 375 neoplastic transformation, 320 Mineralocorticoids, 430, 438, 440, 441
Leydig cells, 451 plasma cell dyscrasias, 324 Miscarriage. See also Spontaneous abortion
Li-Fraumeni syndrome, 418 plasma cell myeloma, 324–326 chromosomal abnormalities, 409, 410
Lipase, 372 Waldenström macroglobulinemia, 326 Misinterpretation, of laboratory test, 7
Lipoprotein electrophoresis, 14 Lymphoma, 322 Mixed connective tissue disease
Liquid chromatography/mass spectrometry Lysosomal storage diseases (MCTD), 70
(LC-MS), 52 diagnosis, 193 diagnosis, 70
newborn screening, 52 selected, 194 features, 69
Lithium, 161, 166, 169, 427 laboratory evaluation, 70
Liver diseases Modification of diet in renal disease
acute phase reactants, 364 M (MDRD), 388, 389
laboratory tests Macroamylasemia, 373 Molecular techniques, in
acute (fulminant) hepatic failure, 367, Major histocompatibility complex immunohematology, 302
368–369 (MHC), 83 applications, 302–303
INDEX 477
Monoclonal gammopathies, of unknown Neuroblastoma, 194–195 P

significance, 327 characteristic, 194
Paget disease of bone. See Osteitis deformans
Monoclonal immunoglobulins, 73, diagnosis, 195
Pain management, 170
324–326, 392 Neuropathy, 73
acetaminophen, 170, 171
immunofixation to identify, 17 Neutrophils, 319
aspirin, 171–172
Monocytes, 319, 330 Next-generation sequencing, 60
buprenorphine, 172
MPGS. See Massively parallel genomic NK-cell, 77
methadone, 172
sequencing NKF stresses, 389
PAMPs. See Pathogen associated molecular
Multidrug resistance protein 2 NOA. See Nonobstructive azoospermia
patterns
(MRP2), 363 Nocardiosis, 133
Pancreatic adenocarcinoma, 363
Multiple drug resistance (MDR) transporter Nonalcoholic fatty liver (NAFL), 360
Pancreatic carcinoma, 5, 361, 375
proteins, 361 Nonalcoholic steatohepatitis
laboratory evaluation, 372
Muscle biopsy, 68 (NASH), 360
Pancreatic disorders, 371–382
Mutations, 56 Non-HDL cholesterol, 200
Pancreatic tumors, 375–376
Mycobacterial infections, 122 non-Hodgkin lymphoma, 65
endocrine neoplasms, 375–376
Myelodysplastic/myeloproliferative Nonimmunologic reactions, 307
exocrine neoplasms, 375
neoplasms, 333, 336–337 Nonobstructive azoospermia (NOA), 402
Pancreatitis. See Acute pancreatitis; Chronic
chronic myeloid leukemia, 333–334 Nonseminomatous germ cell tumors
pancreatitis
essential thrombocythemia, 335 (NSGCT), 399, 400
Para-aminohippurate (PAH), 388
polycythemia vera, 334 Nonspecific esterase (NSE), 330
Parasitic infections of blood, 102
primary myelofibrosis, 335 Nonsteroidal anti-inflammatory drugs
nematodes, 102
Myelodysplastic syndromes, 335 (NSAIDs), 319
vector-borne parasites, 102
WHO classification, 336 Normal body flora, 99
Parathyroid glands, 444–447
Myeloid disorders, 328 Novel biomarkers for heart failure risk
Parathyroid hormone (PTH), 77, 359, 387,
acute myeloid leukemia, 328–332 assessment, 218
396, 406, 445
biphenotypic leukemias, 331 NP. See Natriuretic peptides
Paroxysmal nocturnal hemoglobinuria
genetic abnormalities, leukemias, NSGCT. See Nonseminomatous germ cell
(PNH), 78, 226, 247
328–329 tumors
Partial thromboplastin time, 9
mixed lineage leukemias, 331 NT-proBNP assays, 217
Parvovirus B19, 103, 105–106
Myeloid neoplasms, WHO Nuchal translucency (NT), 188, 189, 407
cause of, 106
classification, 329 Nucleic acid amplification, 97
diagnosis, 106
Myeloperoxidase deficiency, 337 5ʹ-Nucleotidase (5ʹ-NT), 240, 358, 359
transmitted by, 105
Myeloproliferative neoplasms, 333 Nutritional supplement, 388
Pathogen associated molecular patterns
Myocardial infarction, 401
(PAMPs), 61
acute, criteria for diagnosis of, 211
troponin as marker for (See Troponin) O Patients
general clinical approach with an
types, universal classification of, 212 Obstructive azoospermia (OA), 402
infectious disease, 95
OGTT. See Oral glucose tolerance test
preparation for laboratory testing, 8
Oligozoospermia, 402, 403
N Oral glucose tolerance test (OGTT),
PE. See Pulmonary embolism
PEP. See Protein electrophoresis
NAFL. See Nonalcoholic fatty liver 378, 382
Peptic ulcer disease, 352, 375, 447
NAIT. See Neonatal alloimmune Osmotic fragility test, 251
Peripheral blood smear
thrombocytopenia Osteitis deformans, 451
acanthocytes, 227
NASH. See Nonalcoholic steatohepatitis Osteomalacia, 450–451
basophilic stippling, 234
Nasopharyngeal carcinoma, 104 Osteomyelitis, 112
dacrocytes, 227
Natriuretic peptides (NP), 216–218 clinical presentation, 112–113
echinocyte, 227
N-benzoyl-l-tyrosyl-p-aminobenzoic diagnosis, 113
elliptocyte, 229
acid, 374 organisms associated with, 113
hemoglobin C disease patient, 228
Negative test, definition of predictive populations at risk for, 112–113
Howell-Jolly body, 228, 231
value, 5 Osteoporosis, 71, 353, 434, 446, 449–450
hypersegmented neutrophils patient, 230
Neisseria gonorrhoeae, 149, 152 Ovarian cancer syndrome, 417–418
macroovalocytes patient, 230
Neonatal alloimmune thrombocytopenia BRCA1/BRCA2 mutations, 417
megaloblastic anemia patient, 230
(NAIT), 274 hereditary, 417
reticulocyte, 231
laboratory evaluation, 275 Ovaries, 408
schistocytes, 232
Neonatal screening, 186 endocrinologic disorders, 455–457
sickle cells, 232
Neonatal testing, 186–187 granulosa cells, 452
spherocytes, 233
Neoplasias, 396 laboratory tests, 455
stomatocytes patient, 231
Neoplastic disorders, 464 physiology, 454–455
target cells, 233, 234
carcinoid tumors, 465 Ovaries function, 454
thalassemia, 234
multiple endocrine neoplasia, 464 female physiology and biochemistry,
Wright’s stain, 231
Neoplastic proliferation, of WBCs, 320 454–455
Peripheral blood smear analysis, 27
Nephelometry, 19, 73 Oxcarbazepine, 161, 166
Peutz-Jeghers syndrome, 419
Nephrolithiasis, 395 Oxycodone, 162, 172, 173, 177
Pheochromocytoma, 203, 204, 442–443, 464
Neural tube defects (NTDs), 407 Oxymoprhone, 173, 177
478 INDEX
Phosphate, 396 Preeclampsia, 409, 410–411, 426, 429 R

Phosphatidylglycerol (PG), 188 Pregnancy
RA. See Rheumatoid arthritis
Photopheresis, 314 complication, 409
Radioimmunoassay (RIA), 14
Pituitary gland, disorders related to, 457 ectopic, 408–409
Range
antidiuretic hormone (ADH)/posterior effects on laboratory tests, 406
desirable, 2
pituitary, 461–464 fatty liver of, 411
reference, 2
growth hormone/anterior pituitary, normal, 405–407
therapeutic, 2
457–460 routine testing, 406
Raynaud phenomenon, 69
prolactin/anterior pituitary, 460–461 Pregnancy-associated plasma protein-A
RBC. See Red blood cells (RBC)
Plasma cell dyscrasias, 324 (PAPP-A), 407
RDS. See Respiratory distress syndrome
Plasma cell myeloma, 324 Prematurity, 187
Recurrent spontaneous abortion, 409–410
Plasma markers, of cell injury, 10 cause of, 187
Red blood cells (RBC), 27, 29, 35, 221, 303
Plasmapheresis, 314 defined, 187
alloantibody, 300
Plasmodium falciparum, 102, 103 diagnosis, 187–188
anemia, 221–251
Plasmodium malariae, 102 Prenatal testing, and screening, 186
antigens, 298, 300
Platelet aggregation, 35 Prevalence
compatibility, 297
Platelets, 304 and incidence, difference between, 6
crossmatch, 301
adhesion, 275 Primary hyperparathyroidism, 447–448
erythrocytosis, 248–249
aggregation, 35, 254, 280, 288 Primary myelofibrosis, 335
exchange, 314 (See also
apheresis, 296 Primary sclerosing cholangitis (PSC), 363
Erythrocytapheresis)
count, 271, 335 Proenzymes, 255
morphologic findings, 226
drug-induced defects, 281 Progesterone, 283, 405, 407, 431,
polycythemia vera, 248–249, 249
dysfunction, 387 454–456, 455
senescence, 361
plug formation, 254–255 Prolactin (PRL), 412, 456, 457, 460, 461
Red cell indices, 249
qualitative platelet disorders, 259, 279 Prostate cancer
Reference range, 2
quantitative platelet disorders, 259 malignancy of, 397
Renal azotemia, 386
transfusions, 87, 294, 304, 307 prostate-specific antigen (See Prostate-
Renal disease
Platelet storage pool disease, 278–280 specific antigen (PSA))
chronic renal failure, 386
laboratory evaluation, 280 serum tumor markers, clinical utility
clinical laboratory parameters, 387
syndromes, 279 of, 398
acute kidney injury, novel biomarkers
Pleural fluid analysis, 343–344, 344 Prostate-specific antigen (PSA), 398–399, 403
of, 396
Plumbism. See Lead poisoning benign prostatic hyperplasia (BPH), 398
blood urea nitrogen, 390
PM. See Polymyositis prostate carcinoma, 398
creatinine, 387–388
Pneumocystis jirovecii pneumonia, 134 Protein electrophoresis (PEP), 16, 325, 326,
creatinine ratio, 390
PNH. See Paroxysmal nocturnal 392, 393
cystatin C, evaluation of, 395–396
hemoglobinuria Proteinuria, 74, 204, 390–392, 394, 410, 411
glomerular filtration rate, 388–389
POCT. See Point-of-care test patterns of, 392
proteinuria, patterns, 392
Point-of-care test (POCT) Prothrombin (factor II) deficiency, 262
sodium fractional excretion, 392–393
glucose testing, 53, 379 Protozoal infections, 140–141
tubular dysfunction, 392–393
immunoassay testing on test strip, 54 diagnosis, 141
urea/blood urea nitrogen, 389–390
Polycythemia vera, 248, 334 intestinal protozoa, 140–141
urinalysis, 393–395
diagnosis of, 334–335 PSC. See Primary sclerosing cholangitis
urine protein quantitation, 390–392
Polymerase chain reaction (PCR), 86, Pseudohypoparathyroidism, 449
findings, 385–386
101–103, 109, 120, 126, 133, 154, Pseudomonas species, 74
renal azotemia, 386
332, 334 PT and PTT assays, 31
renal function assessment, 386
with restriction enzyme digestion for PT and PTT mixing studies, 32
selected consequences of untreated renal
detection of mutations, 56 PTH. See Parathyroid hormone
failure, 387
Polymyositis (PM), 67, 68 Pulmonary disease, 339–341
urinary tract infections, 387
autoantibodies and phenotypes, in laboratory tests useful in diagnosis/
Renal ischemia, 67
inflammatory myositis, 69 management of, 345, 345–346
Renal plasma flow (RPF), 388
characteristic feature, 68 Pulmonary embolism (PE), 197, 198, 206,
Respiratory disease, major causes, 340
fulfill clinical diagnostic criteria, 68 207, 340, 342, 345
Respiratory distress syndrome, 347
Positive test Pulmonary fibrosis, 69
acute respiratory distress syndrome
definition of predictive value of, 5 Pulmonary function tests, 346
(ARDS), 347
Postrenal azotemia, 386 Pulmonary interstitial fibrosis, 68
neonatal respiratory distress
Posttransfusion purpura, 273–274 Pure red cell aplasia, 247–248
syndrome, 347
characterization, 273 Pyelonephritis, 385, 386, 394
Respiratory distress syndrome (RDS), 339
laboratory evaluation, 274 and urinary tract infections, 146–147
Respiratory tract infections, 128–130
Precision, 6 Pyruvate kinase (PK) deficiency, 225, 226, 246
Respiratory virus infections, 137
vs. accuracy, 6 Pyuria, 107, 147, 204, 386, 394
Reticulocyte counting, 250
Precursor B- and T-cell neoplasms, 323
Retinopathy, 379
Predictive values
of negative test, 5 Q Rheumatoid arthritis (RA), 65, 70
criteria for diagnosis, 70
of positive test, 5 Quantitative platelet disorders, 259, 260
laboratory evaluation, 71
INDEX 479
Rh typing, 299 Sex hormone-binding globulin (SHBG), 401 diagnosis, 64

Rickettsial diseases Sex steroids, 430, 431, 434, 439, 440, 441 laboratory evaluation, 65
and selected disorders caused by related Sexually transmitted diseases, 149 Systemic mycotic infections, clinical
organisms, evaluation, 101 chlamydial infections, 152–153 findings associated with, 135, 136
Rickettsia rickettsii, 100 gonorrhea, 150, 152 Systemic scleroderma
Rocky Mountain spotted fever (RMSF), 100 herpes simplex virus infections, 153–155 autoantigens and phenotypes, 68
Rotor syndrome, 363 syphilis, 149–150 characterization pathologically, 67
Rubella Sicca syndrome, 65 clinical subtypes, 67
clinical presentation of, 123 Sickle cell anemia, 241–242 diagnosis, 67
diagnosis, 123 and other hemoglobinopathies, 241 laboratory evaluation, 67
laboratory evaluation for, 124 Sickle cell screening assay, 28 Systemic sclerosis, 65, 67
Rubeola Sideroblastic anemia, 247 autoantigens and phenotypes, 68
clinical presentation of, 123 Single-nucleotide polymorphism (SNP) characterization pathologically, 67
diagnosis, 123 identification by array, 59 clinical subtypes, 67
laboratory evaluation for, 124 Sipple syndrome, 464 diagnosis, 67
Sjogren syndrome (SS), 64 laboratory evaluation, 67
diagnostic features, 66
S laboratory evaluation, 66
SA. See Semen analysis (SA) Skin infection T
Schirmer test, for eyes, 66 acute bacterial infections, 115, 118 Tacrolimus, 161, 166, 167
Screening tests for sickle hemoglobin, 251 bacillary angiomatosis, 120 Tangier disease, 202
Secondary hyperparathyroidism, 448 cat-scratch disease, 120 T-cell, 61, 77
Selected commonly monitored drugs. See clinical manifestations, 115 Testes
also Therapeutic drug monitoring fungal infections, 120–121 disorders, 402, 453
antibiotics, 167 lyme disease, 118–120 laboratory tests, 452–453
antidepressants, 169 viral infections with prominent skin Leydig cells, 452
antiepileptics, 168 manifestations, 121 physiology, 451–452
cardiac drugs, 170 measles (rubeola) and rubella, 123 removal, 399
pain management, 170 varicella zoster viral infection, 121, 123 sertoli cells, 451
Selective IgA deficiency, 76 Small format genotyping, 58 Testicular cancer
clinical presentation, 76 SNP. See Single-nucleotide polymorphism alpha-fetoprotein (AFP), 400
diagnosis, 76 Somatostatinomas, 376 germ cell tumors, 399, 400
Semen analysis (SA), 403 Specificity human chorionic gonadotropin
interpretation, 403 formula, for calculation, 4 (hCG), 400
normal values of, 402 of laboratory test, definition of, 4 lactate dehydrogenase (LD), 400
tests, 403 Specimen partial androgen deficiency in
Semen fructose analysis, 403 processing and handling, 9 men, 400
Sensitivity importance of turnaround time, 9 serum tumor markers, clinical utility
definition, 4 timing of blood collection, 9 of, 398
formula for calculation, 4 tubes for blood collection, 9, 10 Testing of donated blood, 296
of laboratory test, definition, 4 Spectrophotometry, 44 Testosterone, 7, 400, 401, 434, 451, 452,
Sepsis and bloodstream infections, 98 assays measuring concentration by, 44 454, 457
bacteremia, 98–99 Spermatozoa, 402 Test results
fungemia, 101–102 Spermatozoa, absence, 402 differences in results
infections caused by Rickettsia, Ehrlichia, Sperm morphology, 403 between samples of venous, arterial,
and Anaplasma, 100–101 Spontaneous abortion, 409–410 and capillary blood, 8
parasitic infections, 102 Staphylococcus aureus, 74 impact of drugs on, 8
babesiosis, 102–103 Streptococcus agalactiae, 98 preanalytical variables affecting, 7
cytomegalovirus, 104–105 Streptococcus pneumoniae, 74, 98 Test selection guidelines, 9
infectious mononucleosis/epstein–barr Streptococcus pyogenes, 98 danger of ordering too many laboratory
virus, 104 Stroke, 401 tests, 9
malaria, 102 Strongyloides stercoralis, 141 use of screening tests before esoteric
parvovirus B19, 105–106 Susceptibility testing, 97 tests, 9
viral infections, 103 Syphilis, 149–150 Thalassemia, 238–239
Serology, 11, 74, 98, 101, 135, 145, 190, 353 causes of, 149–150 clinical causes, 238
of infectious disease, 11 diagnosis, 150 diagnosis, 238, 239
Serotonin (5-hydroxytryptamine), 465 laboratory evaluation for, 151 syndromes, 238
Sertoli-only syndrome, 402 transmission, 150 Therapeutic apheresis, 312
Serum amyloid A (SAA), 72 Systemic autoimmune diseases, 62, 64, 244 cytapheresis, 314
Serum protein electrophoresis gel, 393 Systemic lupus erythematosus, 64 erythrocytapheresis, 314
Severe combined immunodeficiency autoantibodies and clinical associations indications, 312–313, 313
(SCID) syndrome, 77 in, 65 categories, 312
characterization, 77 candidate genes associated with, 64 photopheresis, 314
classification, 77 characteristic, 64 plasmapheresis, 293, 295, 314
480 INDEX
Therapeutic drug monitoring, 160 Trichomonas vaginalis, 148 Viral hepatitis, 365–366
commonly monitored drugs, 161 Troponin, 212–213 acute hepatitis B virus infection, 365
aminoglycosides, 167 analytical methods for measuring, acute hepatitis C virus infection, 365, 366
carbamazepine, 168 213–214 acute hepatitis D virus infection, 365
citalopram, 169 elevation of values, cause of, 211 diagnosis of, 366
cyclosporin, 167 high-sensitivity assays, 215–216 hepatitis serologic tests, 365
digoxin, 170 as marker for myocardial infarction, 214 Viral infections
fluoxetine, 169 role in risk outcomes assessment, 214–215 of blood, 103
fluvoxamine, 169 testing, serial, 215 with prominent skin manifestations, 121
lithium, 169 TSH. See Thyroid-stimulating hormone Viral meningitis, acute, 110
methotrexate, 167 TTR. See Transthyretin Viruses inducing gastroenteritis, 137–138
norfluoxetine, 169 Tuberculosis, 130 Vitamin B12 deficiency, 239
paroxetine, 169 clinical features of, 130 causes, 240
phenobarbital, 168 diagnosis, 131 diagnosis, 240
phenytoin, 168 laboratory evaluation for, 132 Vitamin D deficiency, 359, 446, 448, 449
primidone, 168 Tumor necrosis factor (TNF), 64, 92, 135 Vitamin K deficiency, 268
quetiapine, 169 Turnaround time, 9 laboratory evaluation, 269
sertraline, 169 for assay, 14 VLDL and chylomicrons [CM]-associated
sirolimus, 167 assessment of, 14 abnormalities, 202
tacrolimus, 167 Type III hyperlipoproteinemia, 202 von Willebrand disease, 275
trazodone, 170 classification, 276
tricyclic antidepressants, 169 diagnosis, 277
valproic acid, 168 U laboratory evaluation, 277
vancomycin, 167–168 UDP-glucuronyl transferase, 361, 363 types, subtypes, and expected test results,
venlafaxine, 170 Unconjugated hyperbilirubinemia 277–278
indications for, 161, 162 with hemolysis, 362 von Willebrand factor, 10
laboratory methods, 165 without hemolysis, 363 assays, 34
pharmacokinetic principles, 162 Uracil-DNA glycolase (UNG), 76 major roles, 275
distribution, 163–164 Urea, 389–390
drug absorption, 163 Uremia, 386
drug elimination, 164 Uric acid, 396 W
drug interactions, and dose Urinalysis, 46, 393–395 Waldenström macroglobulinemia, 326
adjustments, 165 stones/crystals, 395 Warfarin (coumadin), 2, 8, 259, 266, 287,
drug metabolism, 164 Urinary casts, 394 288, 304
liberation and routes of drug Urinary-free cortisol (UFC), 380 WDHHA syndrome, 376
administration, 162–163 Urinary tract infections (UTIs), 92, 146 Wermer syndrome, 464
specimen collection, 165 categories of, 146 Western blot, 42
Therapeutic range, 2 diagnosis, 147 White blood cell (WBC)
Thrombotic disorders, 281 laboratory evaluation for, 147 disorder, approach to patient with, 318
anticoagulant therapies, 287–288 symptoms, 147 disorders associated with impaired
laboratory monitoring of, 288 Urinary urobilinogen, 395 function, 337
antiphospholipid antibodies, 284–285 Urine amylase, 373 Chediak–Higashi syndrome, 337
antiplatelet therapies, 288 Urine pH, 394 chronic granulomatous disease, 337
hemolytic–uremic syndrome, 286–287 Urine protein quantitation, 390–392 myeloperoxidase deficiency, 337
hypercoagulable states, 281, 283–284 Urine specific gravity, 395 neoplastic proliferation, 320
diagnosis, 283–284 Urolithiasis, 395 nonneoplastic proliferation, 319
laboratory evaluation, 282 UTIs. See Urinary tract infections Wilson disease, 360
thrombotic thrombocytopenic purpura, Wiskott-Aldrich syndrome, 279, 280, 306
285–286 Women amenorrhea, laboratory evaluation
laboratory evaluation, 286 V of, 457
Thrombotic thrombocytopenic purpura, 285 Vaginitis, 142, 148, 149
Thyroid-stimulating hormone (TSH), 204, Valproic acid, 161, 166, 168, 169, 284
407, 422, 423, 425–430, 426, 457 Vancomycin, 167, 301, 307 X
Thyroid tumors, 429 Vanillylmandelic acid (VMA), 195 X-linked agammaglobulinemia (XLA), 74
Tick (Ixodes), 102 Varicella zoster viral infection, 103, 121, 123 laboratory evaluation, 75
Toll-like receptors, 61 clinical manifestations, 121
Total cholesterol, 199, 200 diagnosis, 123
Transfusion, indications for, 303 evaluation for, 123 Y
cryoprecipitate, 304 Vasculitis, 198 Yersinia enterocolitic, 93, 139, 309
fresh frozen plasma, 304 clinical presentation of, 204–205
platelets, 304 definition, 204
red blood cells, 303–304 diagnosis, 205 Z
Transfusion reaction workup, 309–310 laboratory evaluation for, 205 Zinc protoporphyrin (ZPP), 181, 237, 240
Transthyretin (TTR), 71, 72, 364, 393, Vasectomy, 402 Zollinger–Ellison syndrome, 375
422, 423 Vasoactive intestinal peptide (VIP), 375, 376 Zymogens. See Proenzymes

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Laboratory Medicine Diagnosis of Disease in Clinical Laboratory

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Laboratory Medicine

New York Chicago San Francisco Athens Lisbon Madrid

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outlines, and a general introduction

• This edition has been enhanced by coverage

Blood-smear micrographs demonstrate common

426 CHAPTER 22 The Endocrine System

Infantile hypothyroidism TSH high; free T4 low in a newborn or infant

1. Concepts in Laboratory Medicine 1 13. Diseases of White Blood Cells,

Yash P. Agrawal, MD, PhD Jacqueline J. Haas, MD

Daniel E. Sabath, MD, PhD Thomas P. Stricker, MD, PhD

Acetaminophen (therapeutic) Serum, plasma 10-30 μg/mL 6.62 70-200 μmol/L

Acetoacetic acid Serum, plasma <1 mg/dL 0.098 <0.1 mmol/L

Acetone Serum, plasma <2.0 mg/dL 0.172 <0.34 mmol/L

Acetylcholinesterase Red blood cells 5-10 U/mL 1 5-10 U/L

Activated partial thromboplastin Whole blood 25-40 s 1 25-40 s

Adenosine deaminasea Serum 11.5-25.0 U/L 0.017 0.20-0.43 μKat/L

Adrenocorticotropic hormone (ACTH) (see corticotropin)

Alanineb (adult) Plasma 1.87-5.88 mg/dL 112.2 210-661 μmol/day

Alanine aminotransferase Serum 10-40 U/L 1 10-40 U/L

Albuminb Serum 3.5-5.0 g/dL 10 35-50 g/L

Alcohol (see ethanol, isopropanol, methanol)

Alcohol dehydrogenasea Serum <2.8 U/L 0.017 <0.05 μKat/L

Aldolasea,b Serum 1.0-7.5 U/L 0.017 0.02-0.13 μKat/L

Aldosteroneb (upright) Plasma 7-30 ng/dL 0.0277 0.19-0.83 nmol/L

Aldosterone Urine, 24 h 3-20 μg/24 h 2.77 8-55 nmol/day

Alkaline phosphataseb Serum 50-120 U/L 1 50-120 U/L

α1-Acid glycoprotein Serum 50-120 mg/dL 0.01 0.5-1.2 g/L

α2-Macroglobulin Serum 130-300 mg/dL 0.01 1.3-3.0 g/L

Alprazolam (therapeutic) Serum, plasma 10-50 ng/mL 3.24 32-162 nmol/L

Aluminum Serum, plasma <6 ng/mL 37.06 0.0-222.4 nmol/L

Amino acid fractionation

Continued next page—

Amiodarone (therapeutic) Serum, plasma 0.5-2.5 μg/mL 1.55 0.8-3.9 μmol/L

δ-Aminolevulinic acid Urine 1.0-7.0 mg/24 h 7.626 8-53 μmol/day

Amitriptyline (therapeutic) Serum, plasma 80-250 ng/mL 3.61 289-903 nmol/L

Ammonia (as NH3) b

Amobarbital (therapeutic) Serum 1-5 μg/mL 4.42 4-22 μmol/L

Amoxapine (therapeutic) Plasma 200-600 ng/mL 1 200-600 μg/L

Angiotensin I Plasma <25 pg/mL 1 <25 ng/L

Angiotensin II Plasma 10-60 pg/mL 1 10-60 ng/L

Angiotensin-converting Serum 8-52 U/L 0.017 0.14-0.88 μKat/L

Antidiuretic hormone (ADH, Plasma 1-5 pg/mL 0.926 0.9-4.6 pmol/L

α2-Antiplasmin Plasma 80-130 % 0.01 0.8-1.3 Fraction of 1.0

Antithrombin III Plasma 21-30 mg/dL 10 210-300 mg/L

Antithrombin III activity Plasma 80-130 % 0.01 0.8-1.3 Fraction of 1.0

α1-Antitrypsin Serum 80-200 mg/dL 0.01 0.8-2.0 g/L

Arginineb Plasma 0.37-2.40 mg/dL 57.4 21-138 μmol/L

Arsenic (As) Whole blood <23 μg/L 0.0133 <0.31 μmol/L

Ascorbate, ascorbic acid (see vitamin C)

Asparagineb Plasma 0.40-0.91 mg/dL 75.7 30-69 μmol/L

Aspartate aminotransferase Serum 20-48 U/L 0.017 0.34-0.82 μKat/L

Aspartic acidb Plasma <0.3 mg/dL 75.1 <25 μmol/L

Atrial natriuretic hormone Plasma 20-77 pg/mL 1 20-77 ng/L

Barbiturates (see individual drugs; pentobarbital, phenobarbital, thiopental)

Basophils (see complete blood count, white blood cell count)

Benzodiazepines (see individual drugs; alprazolam, chlordiazepoxide, diazepam, lorazepam)

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Beryllium, toxic Urine >20 μg/L 0.111 >2.22 μmol/L

Bicarbonate Plasma 21-28 mEq/L 1 21-28 mmol/L

Bile acids (total) Serum 0.3-2.3 μg/mL 2.448 0.73-5.63 μmol/L

Biotin Whole blood, 200-500 pg/mL 0.0041 0.82-2.05 nmol/L

Bismuth Whole blood 1-12 μg/L 4.785 4.8-57.4 nmol/L

BNP Plasma <100 pg/mL 1 <100 pg/mL